Inhibition of mitogen-activated protein kinase (MAPK) pathway: a selective therapeutic strategy against melanoma

ABSTRACT

Inhibitors of the MAPK pathway, including MEK-directed proteases and small molecule inhibitors, are cytotoxic to human melanoma cells in vitro and in vivo via apoptotic mechanisms. These compounds are used to kill melanoma cells and to treat subjects with melanoma, either alone or in combination with other therapeutic modalities.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention in the field of biochemistry and medicine relatesto methods to kill melanoma cells and treat melanoma tumors in aselective manner using compositions that inhibit the mitogen-activatedprotein kinase (MAPK) pathway.

[0003] 2. Description of the Background Art

[0004] The MAPK pathways are found in, and highly conserved among, alleukaryotes. These pathways play an integral role in the transduction ofvarious extracellular signals into the nucleus. The best-characterizedmammalian pathway, designated Raf-MEK1/2-ERK1/2, includes the MAPKenzymes also known as ERK1 and ERK2, which are phosphorylated andactivated by the dual-specificity kinases that have been termed“MAPK/ERK kinases” (abbreviated variously as MAPKK1 and MAPKK2 or, aswill be used herein, MEK1 and MEK2). The MEK enzymes are in turnphosphorylated and activated by the Raf kinases (Lewis, TS. et al., AdvCanc Res, 74:49-139 (1998)).

[0005] The MAPK pathway is involved in the regulation of cell growth,survival, and differentiation (Lewis et al., supra). Furthermore,activated MAPK and/or elevated level of MAPK expression have beendetected in a variety of human tumors (Hoshino, R. et al., Oncogene18:813-822 (1999); Salh, B et al., Anticancer Res. 19:741-48 (1999);Sivaraman, V S et al., J. Clin. Invest. 99:1478-483 (1997); Mandell, J Wet al., Am. J. Pathol. 153:1411-23 (1998); Licato, L. L. et al.Digestive Diseases and Sciences 43, 1454-1464 (1998)) and may beassociated with invasive, metastatic and angiogenic activities of tumorcells. Thus, inappropriate activation of the MAPK pathway is anessential feature common to many types of tumors. For this reason,participants in this signaling pathway, such as MEK, are potentialtargets for cancer therapy.

[0006] However, it has generally been observed that inhibitors of signaltransduction, including of the MAPK pathway, are cytostatic in nature,merely arresting the growth of tumor cells but not killing them,creating an expectation that non-traditional approaches would berequired to develop such agents into clinical therapeutics.

[0007] The present inventors and their colleagues observed in theNational Cancer Institute's Antineoplastic Drug Screen (NCI-ADS)database (Koo, H. -M. et al., Canc Res 56:5211-5216 (1996); Monks, A. etal. J Natl Cane Inst 83:757-766 (1991); Grever, M. R. et al., Sem Oncol19:622-638 (1992)) that the lethal factor (LF) of Bacillus anthracis, aMEK-directed protease (Duesbery, N. S. et al., Science 280:734-737(1998); Vitale, G. et al., Biochem Biophys Res Comm 248:706-711 (1998))and the small molecule pharmacological MEK inhibitor PD98059 (Dudley, D.T. et al., Proc Nat'l Acad Sci USA 92:7686-7689 (1995); Alessi, D. R. etal., J Biol Chem 270:27489-27494 (1995)) displayed enhanced growthinhibition specifically against melanoma lines among the many differenttumor cell lines tested. Another small molecule MEK inhibitor, PD184352,has been described (Sebolt-Leopold, J. S. et al., Nature Med. 5: 810-816(1999)).

[0008] The present invention is based on subsequent work wherein theinventors explored the mechanism by which inhibition of the MAPKsignaling would selectively inhibit the growth of the human melanomacells.

[0009] The incidence of malignant melanoma is increasing dramatically inall parts of the world (Parker, S. et al., CA Cancer J Clin 47:5-27(1997)). The estimated lifetime risk for the population withlight-colored skin is reaching 1 in 75 by the year 2000. Melanoma can beone of the most aggressive malignancies as the tumor readilymetastasizes early in disease progression. Early detection and surgicalintervention remain the mainstays of treatment for localized melanoma.Despite improved diagnosis and characterization, long-term survival fromthe advanced disease has not improved.

[0010] The present ‘inventors’ observation that inhibition of MAPKsignaling specifically triggers an apoptotic response in human melanomacells, but not in normal melanocytes or in other cell types, provides anovel, selective therapeutic strategy for systematic treatment ofmalignant melanomas.

[0011] Citation of the above documents is not intended as an admissionthat any of the foregoing is pertinent prior art. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicant and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

SUMMARY OF THE INVENTION

[0012] The present inventors found that sustained inhibition of MAPKsignaling in human melanoma cells, preferably at least 72 hours induration, evoked a selective cytotoxic response via apoptosis andresults in killing of the entire cell population so treated. The resultsdescribed herein serve as the basis of a novel, specific therapeuticstrategy for melanoma based on inhibition of the MAPK pathway that leadsto cytotoxicity and death of melanoma cells by apoptosis.

[0013] This invention provides a selective therapeutic approach to treatmelanoma. According to the present approach, inhibition of MEK1/2 whichare essential components of a MAPK (=ERK1/2) pathway, induces apoptosisselectively in melanoma cells, particularly in human melanoma cells,resulting in a cytotoxic rather than a cytostatic effect.

[0014] In one embodiment, a method of killing melanoma cells comprisescontacting the cells for an effective time with an effective amount ofan inhibitor of the MAPK pathway which induces apoptosis in the cells.Preferably the contacting is in vivo.

[0015] In another embodiment, a method of protecting against melanoma ina susceptible subject comprises administering to the subject that is (a)at risk for development of melanoma or (b) in the case of an alreadytreated subject, at risk for recurrence of melanoma, an effective amountof a MAPK pathway inhibitor.

[0016] In another embodiment, a method of inducing an antitumor responsein a mammal having melanoma comprises administering an effective amountof an inhibitor of the MAPK pathway to the mammal, which MAPK pathwayinhibitor is cytotoxic to melanoma cells, thereby inducing an antitumorresponse that is

[0017] (a) a partial antitumor response characterized by

[0018] (i) at least a 50% decrease in the sum of the products of maximalperpendicular diameters of all measurable lesions;

[0019] (ii) no evidence of new lesions, and

[0020] (iii) no progression of any preexisting lesions, or

[0021] (b) a complete antitumor response characterized by thedisappearance of all evidence of melanoma disease for at least onemonth.

[0022] In another embodiment, a method of inhibiting growth or recurrentgrowth of a melanoma tumor in a mammal having melanoma or at risk formelanoma growth or recurrence, comprises administering an effectiveamount of an inhibitor of the MAPK pathway to the mammal, therebyinducing a cytotoxic response leading to apoptosis of melanoma cells inthe mammal, which inhibits the growth or recurrent growth of themelanoma tumor.

[0023] In the above embodiment, the MAPK pathway inhibitor is preferablyan inhibitor of MEK (i.e., MEK1 and MEK2) such as Bacillus anthracislethal factor (LF), a functional derivative thereof or anotherMEK-specific protease that results in the efficient induction ofapoptosis in human melanoma cells.

[0024] In another embodiment, inhibition of the MAPK pathway is by asmall molecule inhibitor, preferably PD98059 or U0126, which alsoresults in the efficient induction of apoptosis in human melanoma cells.

[0025] In response to MEK inhibition, melanoma cells initiallyexperience G1 cell cycle arrest. However, sustained inhibition of thepathway leads to efficient apoptosis. This differs from the response ofmost other cell types tested so far: these other cells remain arrestedin G1, even after prolonged MEK inhibition, without signs of cell death.Thus, the effect on these other cell types is cytostatic rather thancytotoxic.

[0026] Apoptosis of melanoma cells by MEK inhibition coincides with acomplete inhibition of the activation of MAPK1/2 (=ERK1/2), kinaseenzymes “downstream” from MEK1/2.

[0027] MEK inhibition also stimulates melanoma cells to produce melanin,a phenotype associated with differentiated melanocytes and melanomacells. Cyclic AMP (cAMP)-elevating agents are known to inducedifferentiation accompanied by melanin production in melanoma cells.While the cAMP-elevating agents, such as Bacillus anthracis edema factor(EF) and isobutylmethylxanthine (IBMX) synergize with MEK inhibitors intheir effects on melanin production, EF or IBMX dominantly antagonizeapoptosis induced by MEK inhibition.

[0028] In contrast to its effect on melanoma cells, MEK inhibition, isnot cytotoxic to normal human melanocytes. While MEK inhibitioncompletely blocks the activation of MAPK in normal melanocytes, and thecells are arrested in G1, apoptosis is not detected even after prolongedinhibition.

[0029] Long-term treatment of mixed cultures of melanoma cells and skinkeratinocytes (in a 1:1 ratio) with LF results in selective killing ofthe melanoma cells while the surviving keratinocytes remain reversiblyarrested in G1.

[0030] The present invention is useful as a selective therapeuticstrategy for the treatment of melanoma. The inhibition of MAPK pathwayis cytotoxic (mediated by an apoptotic mechanism) only to melanoma cellswhereas it is cytostatic (growth arrest) to most other cell types andnormal melanocytes. Therefore, such a strategy provides a selective andsystematic method to treat malignant melanoma.

[0031] An important advantage of this invention is the relative absenceor mildness of side effects that accompany this systemic therapeuticstrategy. This is because the inhibiting the MAPK pathway is cytostaticand therefore reversible in most cell types. Hence, minimal side-effectsare expected and those unforeseen side-effects should reverse uponcessation of therapy.

[0032] The approach described herein may be combined with othertreatment modalities to prevent initial appearance, or more importantly,prevent recurrence of melanoma.

[0033] The present invention therefore provides a method of killingmelanoma cells comprising contacting the cells for an effective timewith an effective amount of an inhibitor of the MAPK pathway whichinduces apoptosis in the cells. A preferred inhibitor is a MEK-directedprotease, such as Bacillus anthracis lethal factor (“LF”)or a functionalderivative thereof.

[0034] The inhibitor may be an organic small molecule, such as PD98059,U0126 or PD 184352.

[0035] In the above method, the contacting is preferably in vivo. Thekilling preferably results in measurable regression of melanoma tumor orattenuation of melanoma growth.

[0036] Also provided is a method of protecting against melanoma in asusceptible subject, comprising administering to the subject that is (a)at risk for development of melanoma or, (b)in the case of an alreadytreated subject, at risk for recurrence of melanoma, an effective amountof a MAPK-inhibitor.

[0037] The invention is also directed to a method of inducing anantitumor response in a mammal having melanoma, comprising administeringan effective amount of an inhibitor of the MAPK pathway to the mammal,which inhibitor is cytotoxic to melanoma cells, thereby inducing anantitumor response that is

[0038] (a) a partial antitumor response characterized by

[0039] (i) at least a 50% decrease in the sum of the products of maximalperpendicular diameters of all measurable lesions;

[0040] (ii) no evidence of new lesions, and

[0041] (iii) no progression of any preexisting lesions, or

[0042] (b) a complete antitumor response characterized by thedisappearance of all evidence of melanoma disease for at least onemonth.

[0043] In the foregoing method, the inhibitor is preferably aMEK-directed protease such as LF or a functional derivative thereof.Alternatively, the inhibitor may be an organic small molecule,preferably PD98059, U0126 or PD 184352.

[0044] In the above methods, the mammal is preferably a human.

[0045] This invention also provides a method of inhibiting growth orrecurrent growth of a melanoma tumor in a mammal having melanoma or atrisk for melanoma growth or recurrence, comprising administering aneffective amount of an inhibitor of the MAPK pathway to the mammal,thereby inducing a cytotoxic response leading to apoptosis of melanomacells in the mammal, which inhibits the growth or recurrent growth ofthe melanoma tumor. In the foregoing method, the inhibitor is preferablya MEK-directed protease such as LF or a functional derivative thereof.Alternatively, the inhibitor may be an organic small molecule,preferably PD98059, U0126 or PD 184352.

BRIEF DESCRIPTION OF THE DRAWINGS

[0046]FIGS. 1 and 2 are graphs showing apoptosis induced by inhibitionof the MAPK pathway in human melanoma cells. FIG. 1 shows induction ofapoptosis by the MEK-directed protease LF.

[0047]FIG. 2 shows induction of apoptosis by the small molecule MEKinhibitor PD98059. Apoptosis was quantified by staining DNA with DAPIfollowed by examining nuclear morphology of the stained cells. Standarddeviations (of quadruplicate samples) are indicated

[0048] FIGS. 3A-3G show results of flow cytometric analysis of the cellcycle response of melanoma cells to sustained inhibition of the MAPKpathway. The progressive cell cycle response of MALME-3M cells to MAPKpathway inhibition by LF (FIGS. 3B-3D) or PD98059 (FIGS. 3E-3G) isshown. Percentage apoptosis indicated in each panel was quantifiedindependently by staining a portion of the sample with DAPI followed byexamination of nuclear morphology. Based on the control (0 hr), the 2Cand 4C DNA content are indicated.

[0049]FIG. 4 is an immunoblot of active phospho-MAPK in MALME-3M cellstreated with LF or PD98059 for the indicated durations (hours). Controlswere treated with protective antigen (“PA”) of the anthrax toxin complexalone or with DMSO for 72 hours. As loading control, the blot wasstripped and re-probed with anti-MAPK antibodies (1:1 mixture ofanti-ERK1 and ERK2 antibodies) shown in the lower panel). Resultsindicated equivalent loading of the gels with MAPK.

[0050]FIGS. 5A and 5B show the synergistic effect of EF and IBMX onmelanogenesis induced by LF or PD98059 respectively in UACC-257 cellsand MALME-3M cells. The melanin content was expressed in arbitrary unitsas a ratio compared to controls treated with PA alone (set as 1.0). Theupper photographs (circles) correspond to the bars in the graphs belowand depict a microplate well of UACC-257 cells (seeded at 4×10⁶cells/well). A single inoculation of DMSO (control) or 20 μM PD98059, anon-apoptotic concentration was used.

[0051]FIG. 6 shows the antagonistic effect of EF on apoptosis triggeredby LF in UACC-257 cells.

[0052]FIG. 7 shows active phospho-MAPK immunoblots of the duplicateUACC-257 cells treated as described for FIGS. 5A, 5B and 6. Loadingcontrols were performed as described for FIG. 4

[0053]FIG. 8 shows the antagonistic effect of EF on apoptosis triggeredby LF and the antagonistic effect of IBMX on apoptosis triggered byPD98059 in MALME-3M cells

[0054]FIG. 9 shows active phospho-MAPK immunoblots of the duplicateMALME-3M cells treated as described for FIGS. 5A, 5B and 8. Loadingcontrols were performed as described for FIG. 4.

[0055]FIG. 10 is an immunoblot and FIG. 11A-1D are cytograms showingresponses of normal human primary melanocytes to inhibition of the MAPKpathway by LF. FIG. 10 shows an active phospho-MAPK immunoblot (top) andtotal MAPK immunoblot (bottom) of normal melanocytes (NHEM) or melanomacells (MALME-3M) treated with PA alone (“PA”) or LF plus PA (“LF”) forthe indicated durations. FIGS. 11A-11B show cell cycle profiles of NHEMcells treated with “PA” or “LF” for 96 hours. FIGS. 11C and 11C showprofiles of MALME-3M cells treated in the same way for 72 hours.Percentage apoptosis, measured independently is indicated in the upperright comer of each panel.

[0056] FIGS. 12A-12D are cytograms and FIG. 13 is an immunoblotdemonstrating the effects of MAPK signal inhibition on normal humanmelanocytes. NHEM cells or M14-MEL melanoma cells were cultured inMelanocyte Growth Medium-3 (Clonetics). NHEM cells were treated withPD98059 for 72 hours. M14-MEL melanoma cells were treated either withPD98059 for 48 hours. Controls were treated with DMSO. FIGS. 12A-12D arecell cycle profiles and apoptosis (independently quantified andindicated in the upper right of each panel). The immunoblot (FIG. 13)shows active phospho-MAPK and total MAPK. The MEK inhibitor U0126 alsodid not induce apoptosis in normal melanocytes.

[0057]FIG. 14 is the graph showing in vivo effect of the MEK-directedprotease LF on human melanoma M14-MEL xenografts in athymic nude mice.Results are shown as estimated tumor weights. When tumors reached anaverage of 95 mg in mass, one group (n=6) was treated with PA (6μg/mouse) alone daily for 13 days (d. 13-25) as control. Mice of theother group (n=7) were treated daily, first with 6 μg PA+2 μg LF/mousefor 4 days (Δ: days 13-16) and then wit 6 μg PA+4 μg LF/mouse for theremaining 9 days (▴: days 17-25). See Example I for methods. Tumorweight (line graph), and tumor depth in mm (column graph inset) areshown along with standard deviations.

[0058]FIG. 15 is a graph showing apoptosis induced by the MEK inhibitorU0126 (10 μM, single inoculation). Apoptosis was quantified by stainingDNA with DAPI followed by examining nuclear morphology of the stainedcells. Standard deviations (of quadruplicate samples) are shown.

[0059]FIG. 16A and 16B show in vivo effects of LF treatment on M14-MEL(FIG. 16A) or SK-MEL-28 (FIG. 16B) melanoma xenograft tumor growth. Whenxenograft growth was established, the tumors were treated with 6 μg PA+2μg LF/mouse (PA+LF, ▪) or 6 μg PA/mouse as controls (PA, O) every otherday. Tumor volume (mm³) is shown along with standard deviations. (PanelA) M14-MEL xenograft tumors (˜450 mm³) were treated with 7 doses(d.17-29, indicated by ↑ above x-axis). (Panel B) SK-MEL-28 tumors (˜310mm³) were treated with 5 doses (d.27-35, indicated by ↑ above x axis)and then with an additional dose three days later (d.38, indicated by↓). The LF-treated tumors remained in complete regression for over 4weeks (d.65). Both graphs were plotted in the same scale.

[0060]FIG. 17A-17R: show histological analysis of the M14-MEL (A-I) orSK-MEL-28 (J-R) xenograft tumors treated with PA or LF. For M14-MELtumors treated with LF, both growing (D-F) and regressing (G-I) tumorswere examined. (A, D, G, J, M, P). H&E and TUNEL staining andcounterstaining were as in FIG. 14. Cross-sections of tumors (scale inmm); (B, E, H, K, N, Q). H&E staining of the tumor sections (insets inE, H, N, Q show melanin deposits in higher magnification); (C, F, I, L,O, R) TUNEL staining of the adjacent sections-TUNEL-positive cellsstained dark brown and nuclei (or DNA) stained light blue. TUNELpositivity found in the PA-treated M14-MEL tumor (C) was due toendogenous peroxidase activity of infiltrating leukocytes.

[0061]FIG. 18 shows the effect of LF treatment of SK-MEL-28 humanmelanoma xenografts in nude mice by direct intratumoral injection. Whentumor growth was established (volume of ˜310 mm³ on day 27 afterimplantation), tumors were treated with 6 μg PA+2 μg LF/mouse (PA+LF, ▪)or 6 μg PA/mouse as controls (PA, ◯). Five doses were given at 2-dayintervals (days 27-35, ↑) and then an additional dose three days later(day 38, ↓). Standard deviations (SD) of PA-treated tumors, ranged from48 mm³ (day 27) to 250 mm³ (day52); SD's of PA+LF-treated tumors rangedfrom 56 mm³ (day 27) to 32 mm³ (day 35).

[0062]FIG. 19 is a graph showing an inverse relationship between thetumor size and the antitumor effects of LF treatment. When M14-MELmelanoma xenograft tumor size reached various approximate sizes: 76 mm³(▴Δ, n=4 each), 187 mm³ (♦⋄, n=4), 278 mm³ (◯, n=4), or 443 mm³ (▪□,n=7), treatment by direct intratumoral injection was initiated.Treatment involved 7 doses (↑) at 2-day intervals of 6 μg PA+2 μgLF/mouse (open symbols) or 6 μg PA/mouse as controls (solid symbols).Two of the 278-mm³ tumors (* inside ◯) started to regrow after cessationof treatment. At the end of the treatment interval (day 14), “control”tumors treated with PA showed significant variation in volume (SD from109 to 263 mm³), whereas, only tumors of the 443-mm³ group survivedPA+LF treatment and were of various sizes (SD from 23 to 131 mm³).

[0063]FIG. 20A and 20B are graphs showing the systemic effect of LFtreatment on the growth of MALME-3M melanoma tumors. MALME-3M cells wereinjected subcutaneously (s.c.) into both right and left dorsal upperflank areas of athymic nude mice. When tumor growth was established, theanimals were randomized (n=5) based on the size of the left-side tumor(˜70 mm³). Treatment began by direct intratumoral (IT) injection at 2day intervals only into right-side (“ipsilateral”) tumors of 10 μg PA+2μg LF/mouse (PA+LF, ▪) or 10 μg PA/mouse for controls (PA, ◯).Contralateral (left-side) tumors were left untreated. When ipsilateraltumors shrunk to a size that could not be visualized (in thePA+LF-treated group), at around day 10, the treatment route was changedto ipsilateral s.c. injection approximately where the tumors had been.Each point is the mean of the group; analysis of each individual showedthat two of the contralateral tumors continuously regressed, whereas onegrew back and the remaining two showed signs of regrowth after the16^(th) treatment.

[0064]FIG. 21 shows the effects of LF administered IV treatment onMALME-3M melanoma xenograft tumor growth. Each curve represents onemouse. Three nude mice bearing MALME-3M tumors were injected IV firstwith 2 μg LF/mouse followed one hour later by 20 μg PA/mouse. Treatmentwas either daily (◯and □) or at 2-day intervals (♦). For the dailytreatment animal, a total of 4 doses was given; for the 2-day intervalschedule, a total of 7 doses were given (arrowheads).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] Hyperactivated or constitutively active kinases of the MAPKpathways are found in a variety of human cancers (Mandell et al., supra;Hoshino et al., supra; Sivaraman, et al., supra; Licato, L. L. et al.,supra). These kinases offer potential therapeutic targets for treatmentof the various forms of cancer. However, all known inhibitors of signaltransduction including inhibitors of MAPK signaling have been found tobe only cytostatic toward tumor cells. The present inventors havediscovered that sustained inhibition of MAPK signaling by MEK inhibitorsevokes a cytotoxic response via apoptosis that is selective for melanomacells, particularly human melanoma. Induction of apoptosis isindependent of the differentiation and/or growth arrest process inducedby the MAPK signal inhibition. Furthermore, the cytotoxic response washighly specific to malignant melanoma cells as normal melanocytes didnot undergo apoptosis in response to inhibition of the MAPK pathway.These results serve as the basis for the present invention that targetsthe MAPK pathway as a selectively cytotoxic melanoma-specifictherapeutic strategy.

[0066] The term “sustained,” as used herein in connection withinhibition of, or contact with, tumor cells or tumor tissue, is definedin terms of (a) duration of contact of the agent with the melanoma cellsor (b) the number of repeated cycles of treatment necessary foreffective inhibition of tumor growth, stabilization or reduction oftumor size, or decrease in the number of tumor lesions. In a preferredembodiment, melanoma cells are subjected to an effective concentrationof an inhibitor for at least 48 hours, more preferably 72 hours, evenmore preferably 96 hours to achieve the desired apoptosis and killing ofa sufficient proportion of the cell population to have a discernibleantitumor effect. In vivo, sustained contact is achieved by repeatedadministration of the inhibitor, preferably by injection, for the numberof cycles necessary to result in the apoptotic death of a significantnumber of tumor cells that results in the desired pharmacological orclinical effect.

[0067] In one embodiment, the subject is given one weekly dose of theinhibitor, systemically or intratumorally. More preferably, theinhibitor is given twice per week, thrice per week or even daily. Whilethe number of repeated doses to achieve the desired pharmacologic effectcannot always be predicted precisely in advance, those skilled in theart will know how to make these assessments, adjust doses and routes,change agents, etc., all as part of the routine clinical approach totreating subjects with melanoma.

[0068] Because of the “geometry” of cell death in a growing tumor invivo, the cytotoxic compositions of the present invention may result inan “inside-out” killing of tumor cells after systemic (e.g.,intravenous) or direct intratumoral administration. The mass of cellsdying and dead within the tumor may remain for a period so that tumorshrinkage is not immediately detectable, even though the tumor is beingsuccessfully treated with the cytotoxic therapy of this invention. Abetter clinical end point may be the cessation of progressive growth ofthe tumor or a delay for a period of at least one month, preferably atleast three months, more preferably at least six months.

[0069] Preferably, the treatment is continued until every detectabletumor cell is killed. Alternatively, other clinical endpoints are usefulfor gauging the success of the present compounds and methods, forexample, significant shrinkage of the tumor mass. In any case,injections of the agent into the site formerly occupied by the tumor canbe given intermittently even after the tumor has disappeared, for aperiod of days, weeks or months (see Examples).

Inhibitors of MEK MEK-Directed Proteases

[0070] The term “MEK-directed protease activity” refers the proteolyticactivity of a protease on MEK1 resulting in inactivation of MEK1. Thisterm is intended to include protease activity on any member of the MEKfamily. The designation MEK refers to a family of protein kinases thatare part of the MAPK pathway. Examples are MEK1, MEK2 and MEK3, etc.).These proteins share sequence similarity, particularly at theN-terminus. See, for example, Duesbery, N S et al., CMLS Cell. Mol. LifeSci. 55:1599-1609 (1999).

[0071] Thus, a MEK-directed protease refers to

[0072] (1) a protease acting on members of the MEK protein family,

[0073] (2) a protease that acts on conservative amino acid substitutionvariants or other conservatively modified variants thereof; and

[0074] (3) a protease that acts on allelic or polymorphic variants,muteins and homologues in other species with greater than about 60%,preferably greater than about 70%, more preferably greater than about80%, most preferably greater than about 90% sequence identity to MEK1,MEK2, MEK3, etc.

[0075] In one embodiment, MEK (i. e., MEK1 and MEK2) is inhibited byBacillus anthracis lethal factor (LF), a MEK-specific protease. LF iscytotoxic toward V12 H-ras-transformed NIH 3T3 cells and causesregression of MEK dependent tumor xenografts of these cells (Duesbery etal. Proc. Natl. Acad. Sci. USA 98: 4098-4094).

[0076] In another embodiment, the protease is a Yersinia protein, YopJ,and its homologues in other species and genera (avrRxv, Y4LO, AvrA),proteases that act on MEK1. LF, YopJ and their homologues, functionalderivatives and mimetics are useful for inhibiting the MAPK pathway andhaving a cytotoxic action on melanoma cells and therefore, on melanomatumors in vivo. See co-pending applications PCT US99/07126, filed Mar.31, 1999, and having a priority date of Apr. 1, 1998; and U.S.Provisional Application Ser. No: 60/183,901, filed Feb. 22, 2000, herebyincorporated by reference in their entirety.

[0077] According to the present invention, the anti-melanoma cytotoxicprotease inhibitor (or homologue or mimetic) exerts is proteolyticaction by recognizing a specific amino acid sequence present in MEK1 orin any member of the MEK family. Thus, methods described herein astargeting MEK1 can be carried out similarly without undueexperimentation and with the same expected effect using an inhibitoractive on any other MEK family member.

[0078] While the present disclosure exemplifies use of B. anthracis LFas a MEK pathway inhibitor, it is to be understood that homologues of LFfrom other Bacillus species and mutants thereof that possess thecharacteristics disclosed herein are intended within the scope of thisinvention.

[0079] Also included is a “functional derivative” of LF, which is meansan amino acid substitution variant, a “fragment,” or a “chemicalderivative” of LF, which terms are defined below. A functionalderivative retains at least a portion of the relevant LF activity, thatof proteolysis of MEK1 which permits its utility in accordance with thepresent invention.

[0080] With respect to the use of YopJ from Yersinia pestis or Yersiniapseudotuberculosis, it is to be understood that homologues of YopJ fromother Yersinia species, and mutants thereof, that possess thecharacteristics disclosed herein are intended within the scope of thisinvention. Also included are “functional derivatives” of YopJ (asdescribed above for LF).

[0081] A functional homologue must possess MEK-protease activity. Inview of this functional requirement, use of homologous proteins to LFand YopJ from other bacterial species and genera, as well as from plantor animals sources, including proteins not yet discovered, fall withinthe scope of the invention if these proteins have sequence homology andthe recited biochemical and biological activity.

[0082] It is within the skill in the art to obtain and express such aprotein using DNA probes based on the sequence of LF or YopJ orSalmonella-derived or plant-derived homologues already characterized.Then, the protein's biochemical and biological activity can be testedreadily using art-recognized methods such as those described herein, forexample, a standard gel mobility shift assay for proteolysis of thesubstrate protein MEK1, or inhibition of MEK1-mediated phosphorylationof its natural substrate, MAPK, or of a model substrate. Finally, abiological assay of anti-melanoma activity such as those exemplifiedherein where apoptosis or other measures of cytotoxic action of theprotein are assessed, will indicate whether the homologue has therequisite activity to qualify as a functional homologue.

[0083] A “variant” of the MEK-directed protease refers to a moleculesubstantially identical to either the full protein or to a fragmentthereof in which one or more amino acid residues have been replaced(substitution variant) or which has one or several residues deleted(deletion variant) or added (addition variant). A “fragment” of theMEK-directed protease refers to any subset of the molecule, that is, ashorter polypeptide of the full length protein.

[0084] A preferred group of MEK-directed protease variants are those inwhich at least one amino acid residue and preferably, only one, has beensubstituted by different residue. For a detailed description of proteinchemistry and structure, see Schulz, G E et al., Principles of ProteinStructure, Springer-Verlag, New York, 1978, and Creighton, T. E.,Proteins: Structure and Molecular Properties, W. H. Freeman & Co., SanFrancisco, 1983, which are hereby incorporated by reference. The typesof substitutions that may be made in the protein molecule may be basedon analysis of the frequencies of amino acid changes between ahomologous protein of different species, such as those presented inTable 1-2 of Schulz et al. (supra) and FIG. 3-9 of Creighton (supra).Based on such an analysis, conservative substitutions are defined hereinas exchanges within one of the following five groups: 1 Small aliphatic,nonpolar or slightly Ala, Ser, Thr (Pro, Gly); polar residues 2 Polar,negatively charged residues Asp, Asn, Glu, Gln; and their amides 3Polar, positively charged residues His, Arg, Lys; 4 Large aliphatic,nonpolar residues Met, Leu, Ile, Val (Cys) 5 Large aromatic residuesPhe, Tyr, Trp.

[0085] The three amino acid residues in parentheses above have specialroles in protein architecture. Gly, the only residue lacking a sidechain, imparts flexibility to the chain. Pro, because of its unusualgeometry, tightly constrains the chain. Cys can participate in disulfidebond formation which is important in protein folding.

[0086] More substantial changes in biochemical, functional (orimmunological) properties are made by selecting substitutions that areless conservative, such as between, rather than within, the above fivegroups. Such changes will differ more significantly in their effect onmaintaining (a) the structure of the peptide backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b) thecharge or hydrophobicity of the molecule at the target site, or (c) thebulk of the side chain. Examples of such substitutions are (i)substitution of Gly and/or Pro by another amino acid or deletion orinsertion of Gly or Pro; (ii) substitution of a hydrophilic residue,e.g., Ser or Thr, for (or by) a hydrophobic residue, e.g., Leu, Ile,Phe, Val or Ala; (iii) substitution of a Cys residue for (or by) anyother residue; (iv) substitution of a residue having an electropositiveside chain, e.g., Lys, Arg or His, for (or by) a residue having anelectronegative charge, e.g., Glu or Asp; or (v) substitution of aresidue having a bulky side chain, e.g., Phe, for (or by) a residue nothaving such a side chain, e.g., Gly.

[0087] Most acceptable deletions, insertions and substitutions accordingto the present invention are those that do not produce radical changesin the characteristics of the protein in terms of its proteolyticactivity. However, when it is difficult to predict the exact effect ofthe substitution, deletion or insertion in advance of doing so, oneskilled in the art will appreciate that the effect can be evaluated byroutine screening assays such as those described here, without requiringundue experimentation.

[0088] Whereas shorter chain variants can be made by chemical synthesis,for the present invention, the preferred longer chain variants aretypically made by site-specific mutagenesis of the nucleic acid encodingthe polypeptide, expression of the variant nucleic acid in cell culture,and, optionally, purification of the polypeptide from the cell culture,for example, by immunoaffinity chromatography using specific antibodyimmobilized to a column (to absorb the variant by binding to at leastone epitope).

[0089] The activity of a variant present in a cell lysate or a morehighly purified preparation is screened in a suitable screening assayfor the desired characteristic, preferably the proteolysis of MEK1. Itis also possible to follow the immunological character of the proteinmolecule is assayed by alterations in binding to a given antibody, andmay measured by competitive immunoassay. Biochemical or biologicalactivity is screened in an appropriate assay, as described below.

[0090] A “mimetic” of a MEK-directed protease is an agent, generally apolypeptide or peptide molecule, that recognizes MEK, e.g., MEK1, as asubstrate and cleaves MEK1 at the same site cleaved by full-length,native protease such as LF or YopJ. Thus, such mimetics includehomologues, peptides, conservative substitution variants, as well asdeletion variants that retain the protease active site and proteolyticaction on MEK1. Such mimetics are tested using assays for proteaseactivity, e.g., MEK1 mobility shift assays, MOS-induced activation ofMAPK in oocytes and myelin basic protein (MBP) phosphorylation, asdescribed below. In assessing a mimetic, LF is generally the positivecontrol for protease activity. A mimetic has at least about 25% of theactivity of this positive control, more preferably at least about50-100% of the activity.

[0091] Also useful in the present methods are agents that potentiate orpromote the above proteolytic activity may be used along with LF orYopJ, their homologues or mimetics to promote their anti-melanomaactivity. A “potentiator” of the protease is an agent that activates(promotes, enhances, increases) the proteolytic activity and isidentified by in vitro or in vivo assays of this activity or downstreamactivities in the MAPK pathway.

[0092] Samples that are treated with a candidate protease potentiatorare compared to control samples that have not been treated with the testcompound. This permits assessment of the presence and extent ofactivation of MEK 1 protease activity. Control samples (untreated withtest compounds) are assigned a relative protease activity value of 1.Activation is achieved when the measured protease activity value isabout 1.5, more preferably 2.0 or greater. Potentiatiors can also beevaluated in a cellular assay, for example an assay for growthinhibition or apoptosis of human melanoma cells in culture asexemplified herein.

Fusion Proteins

[0093] The present invention utilizes a fusion protein comprising theMEK-directed protease (or homologue, functional derivative or mimetic)that is fused to another peptide or polypeptide that confers usefulproperties on the fusion protein.

[0094] One protein useful as a fusion partner is the domain of LF thatbinds to the protective antigen (“PA”) of the anthrax toxin complexproduced by Bacillus anthracis (Leppla, SH, “Anthrax Toxins,” In:Handbook of Natural Toxins: Bacterial Toxins and Virulence Factors inDisease, Moss, J. et al., eds., Dekker, New York, 1995). For a recentreview of anthrax toxins, see Duesbery, NS et al., CMLS Cell. Mol LifeSci. 55:1599-1609 (1999). PA is one of three protein components of the“lethal” or “anthrax” toxin produced by B. anthracis. The 83 kDa PAbinds to a cell surface receptor present on almost all vertebrate cells,and its C-terminus is necessary for this binding (Singh, Y et al., J.Biol. Chem. 264:19103-19107 (1989); Novak, J. et al., J. Biol. Chem.267:17186-17193 (1992)). After binding, PA is specifically cleaved by aprotease (e.g. furin, clostripain or trypsin), releasing a 20 kDaN-terminal PA fragment while a 63 kDa C-terminal PA fragment (PA63)remains bound. PA63, also referred to as “processed PA,” contains thereceptor binding site at its C-terminus. PA63 forms a heptamericmembrane-inserted channel which mediates the entry of the two otherprotein components of the complex (LF, and Edema factor, EF) into thecytosol via the endosomal pathway (Gordon et al., Infect. Immun.56:1066-1069 (1988); Milne et al., J. Biol Chem. 269:20607-20612(1994)).

[0095] To promote the uptake and processing of the MEK-directed protease(or homologue, derivative or mimetic), a fusion protein is made betweenthe protease and the 250 amino acid PA-binding domain of LF. This willpromote receptor binding and endosomal targeting of the fusion partner.As used herein, the term “PA” is a PA protein (or functional homologueor derivative) that has its receptor binding site intact and functional.U.S. Pat. Nos. 5,591,631 and 5,677,274 (incorporated by reference intheir entirety) describe PA fusion proteins that target PA to particularcells, such as cancer cells, using, as fusion partners, ligands forreceptors on the targeted cells. In contrast, the present inventionexploits the receptor-binding properties of PA by creating fusionproteins between the MEK-directed protease and the PA-binding domain ofLF. The LF domain can be fused at the N- or C-terminus of the protease.The full length MEK-directed protease is not required in this fusionprotein as long as the domain(s) responsible for the protease activityis (are) present. Such fusion proteins have the advantage offacilitating the uptake of the proteolytic polypeptide into theendosomal compartment and ultimately into the cytoplasm of the cellbeing targeted.

Chemical Modification of the Protein

[0096] A “chemical derivative” of a MEK-directed protease containsadditional chemical moieties not normally a part of the protein.Covalent modifications of the protein are included within the scope ofthis invention. Such modifications may be introduced into the moleculeby reacting targeted amino acid residues with an organic derivatizingagent that is capable of reacting with selected side chains or terminalresidues. Such chemically modified and derivatized moieties may improvethe protein's solubility, absorption, biological half life, and thelike. These changes may eliminate or attenuate undesirable side effectsof the protein in vivo. Moieties capable of mediating such effects aredisclosed, for example, in Remington's Pharmaceutical Sciences, MackPublishing Company, Easton Pa. (Gennaro 18th ed. 1990).

Preparation of Recombinant MEK-directed protease (and MEK1) Proteins

[0097] As described herein, native or recombinant MEK-directed proteaseproteins, their homologues and mimetics are used in the methods of theinvention. MEK1, the target of proteolytic activity, may also beprovided in native or recombinant form for testing. Recombinant proteinsmay be particularly convenient for biochemical assays. MEK-directedprotease homologues and functional derivatives such as substitutionvariants and fusion proteins may be prepared recombinantly forevaluation of their mimetic activity and therapeutic activity.Recombinant proteins are prepared by conventional means which aregenerally described below along with methods for biochemical isolationand purification of the proteins from natural sources.

General Recombinant DNA Methods

[0098] Basic texts disclosing general methods of molecular biology, allof which are incorporated by reference, include: Sambrook, J. et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring HarborPress, Cold Spring Harbor, N.Y., 1989; Ausubel, F. M. et al. CurrentProtocols in Molecular Biology, Vol. 2, Wiley-Interscience, New York,(current edition); Kriegler, Gene Transfer and Expression: A LaboratoryManual (1990); Glover, D. M., ed, DNA Cloning: A Practical Approach,vol. I & II, IRL Press, 1985; Albers, B. et al., Molecular Biology ofthe Cell, 2nd Ed., Garland Publishing, Inc., New York, N.Y. (1989);Watson, J. D. et al., Recombinant DNA, 2^(nd) Ed., Scientific AmericanBooks, New York, 1992; and Old, R W et al., Principles of GeneManipulation: An Introduction to Genetic Engineering, 2^(nd) Ed.,University of California Press, Berkeley, Calif. (1981).

[0099] Unless otherwise indicated, a particular nucleic acid sequenceadditionally encompasses conservative substitution variants thereof(e.g., degenerate codon substitutions) and a complementary sequence. Theterm “nucleic acid” is intended to include a gene, cDNA, mRNA, anoligonucleotide (any polynucleotide). Sizes of nucleic acids are statedeither as kilobases (kb) or base pairs (bp). These are estimates derivedfrom agarose electrophoresis or polyacrylamide gel electrophoresis(PAGE), from sequences of nucleic acids which are determined by the useror published. Protein sizes are stated as molecular mass in kilodaltons(kDa) or as length (number of amino acid residues). Proteins sizes areestimated from PAGE, from sequencing, from presumptive amino acidsequences based on nucleic acid sequence, or from published amino acidsequences.

[0100] Oligonucleotides that are not commercially available may bechemically synthesized (Oligonucleotide Synthesis, N. Gait, ed., CurrentEdition), for example, according to the solid phase phosphoramiditetriester method (Beaucage et al., Tetrahedron Lett. 22:1859-1862 (1981))using an automated synthesizer (Van Devanter et. al., Nucleic Acids Res.12:6159-6168 (1984)). Oligonucleotides are purified by native acrylamidegel electrophoresis or by anion-exchange HPLC (Pearson et al., J.Chromatog. 255:137-149 (1983)). The sequence of a cloned gene or asynthetic oligonucleotide can be verified using the chain terminationmethod for sequencing double-stranded templates (Wallace et al., Gene16:21-26 (1981).

Cloning of Nucleic Acids Encoding MEK-1 Proteases and Other Proteins

[0101] In general, a nucleic acid encoding a MEK1 protease, PA, or ahomologous nucleic acid is cloned starting from cDNA or genomic DNAlibraries or is isolated by polymerase chain reaction (PCR)amplification techniques using oligonucleotide primers. For example, LFis isolated from B. anthracis and YopJ is typically isolated from Y.pestis DNA (genomic or cDNA) libraries. Genes for MEK1 can be clonedfrom mammalian libraries, preferably human libraries. For example, MEK1sequences can be isolated from sarcoma libraries from cells with anactivated MAPK pathway. PA can be cloned from a B. anthracis DNAlibrary.

[0102] Amplification techniques using primers may also be employed toamplify and isolate PA, and MEK1 from DNA or RNA (U.S. Pat. Nos.4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods andApplications (Innis et al, eds, 1990)). PCR and ligase chain reaction(LCR) methods can be used to amplify a nucleic acid sequences directlyfrom mRNA, cDNA or genomic DNA. Degenerate oligonucleotides areroutinely designed to amplify homologues.

Small Molecule Inhibitors of MEK

[0103] Also intended within the scope of this invention are smallorganic molecules that act as inhibitors of MEK. As used herein, “smallmolecules” are organic chemical entities that are not biologicalmacromolecules such as proteins or peptides. The small moleculeinhibitors of MEK generally have a molecular mass of less than about2000 D, preferably less than about 1000 D, more preferably less thanabout 500 D.

[0104] In a preferred embodiment, inhibition of MEK by the smallmolecule inhibitor PD98059 results in the efficient induction ofapoptosis in cells of a human melanoma cell line.

[0105] Other small molecule inhibitors of the MAPK pathway are known tobe, or are expected to be, cytotoxic to melanoma cells. These includethe MEK inhibitors PD184352 (Parke-Davis) (Sebolt-Leopold et al., supra)and U0126 (DuPont) (Favata, M et al., J Biol. Chem. 273:18623-18632(1998)), the p38 kinase inhibitor SB 203580 (Schering-Plough) (Cuenda, Aet al., FEBS Lett. 5 364:229-233 (1995)), and the like.

Pharmaceutical Compositions, Their Formulation and Use

[0106] A pharmaceutical composition according to this inventioncomprises the MEK-directed protease (or functional derivative ormimetic) or the small molecule MEK inhibitor, in a formulation that, assuch, is known in the art.

[0107] Pharmaceutical compositions within the scope of this inventioninclude all compositions wherein the MEK-directed protease or othersmall molecule MEK inhibitor is contained in an amount effective toachieve its intended purpose. While individual needs vary, determinationof optimal ranges of effective amounts of each component is within theskill of the art. Typical dosages comprise 0.1 to 100 mg/kg/body wt,though more preferred dosages are described for certain particular uses,below.

[0108] In addition to the pharmacologically active protein or smallmolecule, the pharmaceutical compositions may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically as is well known in theart. Suitable solutions for administration by injection or orally, maycontain from about 0.01 to 99 percent, active compound(s) together withthe excipient.

[0109] The pharmaceutical preparations of the present invention aremanufactured in a manner which is known, for example, by means ofconventional mixing, granulating, dissolving, or lyophilizing processes.Suitable excipients may include fillers binders, disintegrating agents,auxiliaries and stabilizers, all of which are known in the art. Suitableformulations for parenteral administration include aqueous solutions ofthe proteins in water-soluble form, for example, water-soluble salts. Inaddition, suspensions of the active compounds as appropriate oilyinjection suspensions may be administered. Suitable lipophilic solventsor vehicles include fatty oils, for example, sesame oil, or syntheticfatty acid esters, for example, ethyl oleate or triglycerides. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension.

[0110] The compositions may be in the form of a lyophilized particulatematerial, a sterile or aseptically produced solution, a tablet, anampule, etc. Vehicles, such as water (preferably buffered to aphysiologically acceptable pH, as for example, in phosphate bufferedsaline) or other inert solid or liquid material such as normal saline orvarious buffers may be present. The particular vehicle is not critical,and those skilled in the art will know which vehicle to use for anyparticular utility described herein.

[0111] In general terms, a pharmaceutical composition is prepared bymixing, dissolving, binding or otherwise combining the polymer orpolymeric conjugate of this invention with one or more water-insolubleor water-soluble aqueous or non-aqueous vehicles. If necessary, anothersuitable additive or adjuvant is included. It is imperative that thevehicle, carrier or excipient, as well as the conditions for formulatingthe composition are such that do not adversely affect the biological orpharmaceutical activity of the protein, peptide or small molecule.

Subjects, Treatments Modes and Routes of Administration

[0112] The preferred animal subject of the present invention is amammal. The invention is particularly useful in the treatment of humansubjects. By the term “treating” is intended the administering tosubjects of a pharmaceutical composition comprising a MEK inhibitor(whether a protease or a small molecule inhibitor). Treating includesadministering the agent to subjects at risk for developing melanomaprior to evidence of clinical disease, as well as subjects diagnosedwith melanoma who have not yet been treated or who have been treated byother means, e.g., surgery, conventional chemotherapy, and in whom tumorburden has been reduced even to the level of not being detectable. Thus,due to the melanoma-directed cytotoxic effects of the present methods,this invention is useful in preventing or inhibiting melanoma primarygrowth, recurrent growth or metastatic growth.

[0113] The pharmaceutical compositions of the present invention whereinthe MEK-directed protease or inhibitor is combined with pharmaceuticallyacceptable excipient or carrier, may be administered by any means thatachieve their intended purpose. Amounts and regimens for theadministration of can be determined readily by those with ordinary skillin the clinical art of treating any of the particular diseases.Preferred amounts are described below.

[0114] In general, the present methods include administration byparenteral routes, including subcutaneous (s.c.) intravenous (i.v.),intramuscular, intraperitoneal, intrathecal, transdermal, topical orinhalation routes. A preferred route is by direct intratumoralinjection. Alternatively, or concurrently, administration may be by theoral route. The dosage administered will be dependent upon the age,health, and weight of the recipient, kind of concurrent treatment, ifany, frequency of treatment, and the nature of the effect desired.

[0115] In one treatment approach, the compounds and methods are appliedin conjunction with surgery. Thus, an effective amount of the MEKprotease or small molecule MEK inhibitor is applied directly to the siteof surgical removal of a melanoma mass (whether primary or metastatic).This can be done by injection or “topical” application in an opensurgical site or by injection after closure.

[0116] In a preferred embodiment, the specified amount of a MEK proteaseor inhibitor, preferably about 2-100 μg, is added to about 700 ml ofhuman plasma that is diluted 1:1 with heparinized saline solution atroom temperature. Human IgG in a concentration of 500 μg/dl (in the 700ml total volume) may also be used. The solutions are allowed to standfor about 1 hour at room temperature. The solution container may then beattached directly to an iv infusion line and administered to the subjectat a preferred rate of about 20 ml/min.

[0117] In another embodiment, the pharmaceutical composition is directlyinfused i.v. into a subject. The appropriate amount, preferably about2-100 μg, is added to about 250 ml of heparinized saline solution andinfused iv into patients at a rate of about 20 ml/min.

[0118] In the present method, the composition can be given one time butgenerally is administered six to twelve times (or even more, as iswithin the skill of the art to determine empirically). The treatmentscan be performed daily but are generally carried out every two to threedays or as infrequently as once a week, depending on the beneficial andany toxic effects observed in the subject.

[0119] The pharmaceutical formulation for systemic administrationaccording to the invention may be formulated for enteral, parenteral ortopical administration, and all three types of formulation may be usedsimultaneously to achieve systemic administration of the activeingredient.

[0120] For lung instillation, aerosolized solutions are used. In asprayable aerosol preparations, the active protein or small moleculeagent may be in combination with a solid or liquid inert carriermaterial. This may also be packaged in a squeeze bottle or in admixturewith a pressurized volatile, normally gaseous propellant. The aerosolpreparations can contain solvents, buffers, surfactants, andantioxidants in addition to the protein of the invention.

[0121] For topical application, the therapeutic compounds of the presentinvention may be incorporated into topically applied vehicles such assalves or ointments, as a means for administering the active ingredientdirectly to the affected area. Scarification methods, known from studiesof vaccination, can also be used. The carrier for the active agent maybe either in sprayable or nonsprayable form. Non-sprayable forms can besemi-solid or solid forms comprising a carrier indigenous to topicalapplication and having a dynamic viscosity preferably greater than thatof water. Suitable formulations include, but are not limited to,solution, suspensions, emulsions, creams, ointments, powders, liniments,salves, and the like. If desired, these may be sterilized or mixed withauxiliary agents, e.g., preservatives, stabilizers, wetting agents,buffers, or salts for influencing osmotic pressure and the like.Examples of preferred vehicles for non-sprayable topical preparationsinclude ointment bases, e.g., polyethylene glycol-1000 (PEG-1000);conventional creams such as HEB cream; gels; as well as petroleum jellyand the like.

[0122] Other pharmaceutically acceptable carriers according to thepresent invention are liposomes, pharmaceutical compositions in whichthe active protein is contained either dispersed or variously present incorpuscles consisting of aqueous concentric layers adherent to lipidiclayers. The active protein is preferably present in the aqueous layerand in the lipidic layer, inside or outside, or, in any event, in thenon-homogeneous system generally known as a liposomic suspension.

[0123] The hydrophobic layer, or lipidic layer, generally, but notexclusively, comprises phospholipids such as lecithin and sphingomyelin,steroids such as cholesterol, more or less ionic surface activesubstances such as dicetylphosphate, stearylamine or phosphatidic acid,and/or other materials of a hydrophobic nature.

In Vivo Study of MEK-Directed Proteases and Inhibitors Antitumor Effectsof MEK Pathway Inhibitors in Animal Models of Human Tumors

[0124] The MEK inhibitory agents are tested for therapeutic efficacy inwell established rodent models which are considered to be representativeof a human tumor. The overall approach is described in detail in

[0125] 1. Geran, R. I. et al., “Protocols for Screening Chemical Agentsand Natural Products Against Animal Tumors and Other Biological Systems(Third Edition)”, Canc. Chemother. Reports, Part 3, 3:1-112, and

[0126] 2. Plowman, J. et al., In: B. Teicher, ed., Anticancer DrugDevelopment Guide: Preclinical Screening, Clinical Trials and Approval,Part II: In Vivo Methods, Chapter 6, “Human Tumor Xenograft Models inNCI Drug Development,” Humana Press Inc., Totowa, N.J., 1997.

[0127] Both these references are hereby incorporated by reference intheir entirety.

[0128] I. GENERAL TEST EVALUATION PROCEDURES

[0129] A. Calculation of Mean Survival Time

[0130] Mean survival time is calculated according to the followingformula:

[0131] Mean survival time (days)=$\frac{{\sum S} + {AS}_{({A - 1})} - {\left( {B + 1} \right){NT}}}{S_{({A - 1})} - {NT}}$

Definitions

[0132] Day: Day on which deaths are no longer considered due to drugtoxicity. Example: with treatment starting on Day 1 for survival systems(such as B 16):

[0133] Day A: Day 6.

[0134] Day B: Day beyond which control group survivors are considered“no-takes.” Example: with treatment starting on Day 1. For B 16, Day Bis to be established.

[0135] ΣS: If there are “no-takes” in the treated group, ΣS is the sumfrom Day A through Day B. If there are 25 no “no-takes” in the treatedgroup, ΣS is the sum of daily survivors from Day A onward.

[0136] S_((A−1)): Number of survivors at the end of Day (A−1).

[0137] Example: S_((A−1))=number of survivors on Day 5.

[0138] NT: Number of “no-takes” according to the criteria given inProtocols 7.300 and 11.103.

[0139] B. T/C Computed for all Treated Groups

[0140] T/C is the ratio (expressed as a percent) of the mean survivaltime of the treated group divided by the mean survival time of thecontrol group. Treated group animals surviving beyond Day B, accordingto the chart below, are eliminated from calculations: No. of survivorsin Percent of “no-takes” in treated group beyond Day B control groupConclusion 1 Any percent “no-take” 2 <10 drug inhibition 3 ≧10“no-takes” ≧3  <15 drug inhibitions ≧15 “no-takes”

[0141] Positive control compounds are not considered to have “no-takes”regardless of the number of “no-takes” in the control group. Thus, allsurvivors on Day B are used in the calculation of T/C for the positivecontrol. Surviving animals are evaluated and recorded on the day ofevaluation as “cures” or “no-take.”

Calculation of Median Survival Time

[0142] Median Survival Time is defined as the median day of death for atest or control group. If deaths are arranged in chronological order ofoccurrence (assigning to survivors, on the final day of observation, a“day of death” equal to that day), the median day of death is a dayselected so that one half of the animals died earlier and the other halfdied later or survived. If the total number of animals is odd, themedian day of death is the day that the middle animal in thechronological arrangement died. If the total number of animals is even,the median is the arithmetical mean of the two middle values. Mediansurvival time is computed on the basis of the entire population andthere are no deletion of early deaths or survivors, with the followingexception:

[0143] C. Computation of Median Survival Time From Survivors

[0144] If the total number of animals including survivors (N) is even,the median survival time (days) (X+Y)/2, where X is the earlier day whenthe number of survivors is ≦N/2, and Y is the earliest day when thenumber of survivors ≦(N/2)−1. If N is odd, the median survival time(days) is X.

[0145] D. Computation of Median Survival Time From MortalityDistribution

[0146] If the total number of animals including survivors (N) is even,the median survival time (days) (X+Y)/2, where X is the earliest daywhen the cumulative number of deaths is ≧N/2, and Y is the earliest daywhen the cumulative number of deaths is ≧(N/2)+1. If N is odd, themedian survival time (days) is X. Cures and “No-Takes”: “Cures” and“no-takes” in systems evaluated by median survival time are based uponthe day of evaluation. On the day of evaluation any survivor notconsidered a “no-take” is recorded as a “cure.” Survivors on day ofevaluation are recorded as “cures” or “no-takes,” but not eliminatedfrom the calculation of the median survival time.

[0147] E. Calculation of Approximate Tumor Weight From Measurement ofTumor Diameters with Vernier Calipers

[0148] The use of diameter measurements (with vernier calipers) forestimating treatment effectiveness on local tumor size permits retentionof the animals for lifespan observations. When the tumor is implantedsc, tumor weight is estimated from tumor diameter measurements asfollows. The resultant local tumor is considered a prolate ellipsoidwith one long axis and two short axes. The two short axes are assumed tobe equal. The longest diameter (length) and the shortest diameter(width) are measured with vernier calipers. Assuming specific gravity of˜1.0, and π≈3, the mass (in mg) is calculated by multiplying the lengthof the tumor by the width squared and dividing the product by two. Thus,${{Tumor}\quad {{weight}({mg})}} = {\frac{{{length}({mm})} \times \left( {{width}\lbrack{mm}\rbrack} \right)^{2}}{2}\quad {or}\quad {\left( {L \times W^{2}} \right)/2}}$

[0149] The reporting of tumor weights calculated in this way isacceptable inasmuch as the assumptions result in as much accuracy as theexperimental method warrants.

[0150] F. Calculation of Tumor Diameters

[0151] The effects of a drug on the local tumor diameter may be reporteddirectly as tumor diameters without conversion to tumor weight. Toassess tumor inhibition by comparing the tumor diameters of treatedanimals with the tumor diameters of control animals, the three diametersof a tumor are averaged (the long axis and the two short axes). A tumordiameter T/C of 75% or less indicates activity and a T/C of 75% isapproximately equivalent to a tumor weight T/C of 42%.

[0152] G. Calculation of Mean Tumor Weight From Individual ExcisedTumors

[0153] The mean tumor weight is defined as the sum of the weights ofindividual excised tumors divided by the number of tumors. Thiscalculation is modified according to the rules listed below regarding“no-takes.” Small tumors weighing 39 mg or less in control mice areregarded as “no-takes” and eliminated from the computations. In treatedgroups, such tumors are defined as “no-takes” or as true druginhibitions according to the rules shown in the following Table.

[0154] Positive control compounds are not considered to have “no-takes”regardless of the number of “no-takes” in the control group. Thus, thetumor weights of all surviving animals are used in the calculation ofT/C for the positive control. T/C are computed for all treated groupshaving more than 65% survivors. The T/C is the ratio (expressed as apercent) of the mean tumor weight for treated animals divided by themean tumor weight for control animals. SDs of the mean control tumorweight are computed the factors in a table designed to estimate SD usingthe Percent of small Percent of tumors in “no-takes” in treated group incontrol group Action ≦17 Any percent no-take; not used in calculations18-39 <10 drug inbibition; use in calculations ≧10 no-takes; not used incalculations ≧40 <15 drug inhibition; use in calculations ≧15 Code allnontoxic tests “33”

[0155] estimating factor for SD given the range (difference betweenhighest and lowest observation). Biometrik Tables for Statisticians(Pearson E S, and Hartley H G, eds.) Cambridge Press, vol. 1, table 22,p. 165.

Melanotic Melanoma B16 in C57BL/6 Mice Summary

[0156] Tumor homogenate is implanted ip or s.c. in BDF₁ mice. Treatmentbegins 24 hours after either ip or s.c. implant or is delayed until ans.c. tumor of specified size (usually approximately 400 mg) can bepalpated. Results expressed as a percentage of control survival time.The test compound is administered ip, and the parameter is mean survivaltime. Origin of tumor line: arose spontaneously in 1954 on the skin atthe base of the ear in a C57BL/6 mouse. Handbook on GeneticallyStandardized Jax Mice. Roscoe B. Jackson Memorial Laboratory, BarHarbor, Maine, 1962. See also Ann NY Acad Sci 100, Parts 1 and 2, 1963.

Animals

[0157] Propagation: C57BL/6 mice.

[0158] Testing: C57BL/6 or BDF, (C57BL/6×DBA/2) mice.

[0159] Weight: Within a 3-g weight range, with minimum 18 g for males,17 g for females.

[0160] Sex: One sex used for all test and control groups in oneexperiment.

[0161] Experiment Size: Ten animals per test group. For control groups,the number varies according to the number of test groups.

Tumor Transfer

[0162] Propagation: Implant fragment s.c. by trochar or 12-gauge needleor tumor homogenate (see below) every 10-14 days into axillary regionwith puncture in inguinal region.

[0163] Testing: Excise s.c. tumor on Day 10-14.

[0164] Homogenate: Mix 1 g or tumor with 10 ml of cold balanced saltsolution and homogenize, and implant 0.5 ml of this tumor homogenate ipor sc.

[0165] Fragment: A 25-mg fragment may be implanted sc.

Testing Schedule

[0166] Day 0: Implant tumor. Prepare materials. Run positive control inevery odd-numbered experiment. Record survivors daily.

[0167] Day 1: Weigh and randomize animals. Begin treatment withtherapeutic composition.

[0168] Typically, mice receive the test compound in 0.5 ml saline.Controls receive saline alone. The treatment is given as one dose perweek. Any surviving mice are sacrificed 8 weeks of therapy.

[0169] Day 5: Weigh animals and record.

[0170] Day 60: Kill all survivors and evaluate experiment.

Quality Control

[0171] Acceptable control survival time is 14-22 days. Positive controlcompound is 5-fluorouracil: single dose is 200 mg/kg/injection,intermittent dose is 60 mg/kg/injection, and chronic dose is 20mg/kg/injection. T/C lower limit for positive control compound is ≧135%.Check control deaths, no takes, etc.

Evaluation

[0172] Compute mean animal weight on Days 1 and 5, and at the completionof testing compute T/C for all test groups with >65% survivors on Day 5.A T/C value ≦85% indicates a toxic test. An initial T/C≧125% isconsidered necessary to demonstrate activity. A reproduced T/C≧125% isconsidered worthy of further study. For confirmed activity a therapeuticcomposition should have two multi-dose assays that produce a T/C≧125%.

Metastasis after IV Injection of Tumor Cells

[0173] 10⁵ B16 melanoma cells in 0.3 ml saline are injectedintravenously to C57BL/6 mice. The mice are then treated intravenouslywith the test compound in 0.5 ml saline. Controls receive saline alone.The treatment is given as one dose per week. Mice sacrificed after 4weeks of therapy, the lungs are removed and metastases are enumerated.The following are considered to be significant effects: Parameter % ofControl Response B16 Mean or median survival time >130% B16 metastasisMedian number of metastases  <70%

[0174] Human Tumor Xenograft Models

[0175] The preclinical discovery and development of anticancer drugs asimplemented by the National Cancer Institute (NCI) consists of a seriesof test procedures, data review, and decision steps (Grever, M R, SeminOncol., 19:622-638 (1992)). Test procedures are designed to providecomparative quantitative data, which in turn, permit selection of thebest candidate agents from a given chemical or biological class. Below,we describe human tumor xenograft systems, emphasizing melanomas, thatare currently employed in preclinical drug development.

[0176] Since 1975, the NCI approach to drug discovery involvedprescreening of compounds in the i.p.-implanted murine P388 leukemiamodel (see above), followed by evaluation of selected compounds in apanel of transplantable tumors (Venditti, J. M. et al., In: Garrattini Set al., eds., Adv. Pharmacol and Chemother 2:1-20 (1984)) includinghuman solid tumors. The latter was made possible through the developmentof immunodeficient athymic nude (nu/nu) mice and the transplantationinto these mice of human tumor xenografts (Rygaard, J. et al., ActaPathol. Microbiol Scand. 77:758-760 (1969); Giovanella, G. C. et al., J.Natl Canc. Inst. 51:615-619 (1973)). Studies assessing the metastaticpotential of selected murine and human tumor-cell lines (B16, A-375,LOX-IMVI melanomas, and PC-3 prostate adenocarcinoma) and theirsuitability for experimental drug evaluation supported the importance ofin vivo models derived from the implantation of tumor material inanatomically appropriate host tissues; such models are well suited fordetailed evaluation of compounds that inhibit activity against specifictumor types. Beginning about 1990, the NCI began employing human tumorcell lines for large-scale drug screening ((Boyd, M R, In: DeVita, V Tet al., Cancer: Principles and Practice of Oncology, Updates, vol 3,Philadelphia, Lippinicott, 1989, pp 1-12; B. Teicher, ed., AnticancerDrug Development Guide: Preclinical Screening, Clinical Trials andApproval chapter 2). Cell lines derived from seven cancer types (brain,colon, leukemia, lung, melanoma, ovarian, and renal) were acquired froma wide range of sources, frozen, and subjected to a battery of in vitroand in vivo characterization.

[0177] This approach shifted the screening strategy from“compound-oriented” to “disease-oriented” drug discovery (Boyd, supra).Compounds of identified by the screen, demonstrating disease-specific,differential cytotoxicity such as the anti-melanoma activity of thecompounds described herein, were considered “leads” for furtherpreclinical evaluation. A battery of human tumor xenograft models wascreated to deal with such needs.

[0178] The approach used to establish s.c. xenografts from human tumorcell culture lines (obtained from the NCI tumor repository at Frederick,Md.) is outlined in the schematic diagram below.

[0179] The cryopreserved cell lines are thawed, cultured in RPMI 1640medium supplemented with 10% -heat-inactivated fetal bovine serum, andexpanded until the population is sufficient to yield ≧10⁸ cells. Cellsare harvested and then implanted s.c. into the axillary region of 10athymic nu/nu mice (10⁷ cells/0.5 ml/mouse). Preferred housingconditions for these mice are as follows: mice are housed in sterile,polycarbonate, filter-capped microisolator cages (e.g., from Lab.Products, Inc.), maintained in a barrier facility on 12-h light/darkcycles, and provided with sterilized food and water ad libitum. Theimplanted animals are observed twice weekly for tumor appearance. Growthof the solid tumors is monitored using in situ caliper measurements todetermine tumor mass. Weights (mg) are calculated from measurements (mm)of two perpendicular dimensions (length and width) using the formula fora prolate ellipsoid and assuming a specific gravity of 1.0 g/cm³ (GeranR I et al., Cancer Chemother Rep. 3, Part 3:51(1972)). Fragments ofthese tumors may be subjected to histological, cytochemical, andultrastructural analysis to monitor the characteristics of the in vivomaterial and to compare them with those of the in vitro lines and, wherepossible, with those reported for initial patient tumors (Stinson S F etal., Anticancer Res 12:1035-1054 (1992)). Both in vitro and in vivotumor materials should exhibit characteristics consistent with tissuetype and tumor of origin, though differences in the degree ofdifferentiation between some of the cultured cell lines andcorresponding xenograft materials are not uncommon.

[0180] The initial solid tumors established in mice are maintained byserial passage of 30-40 mg tumor fragments implanted s.c. near theaxilla. Xenografts are generally not utilized for drug evaluation untilthe volume-doubling time has stabilized, usually around the fourth orfifth passage. The doubling time of xenografts derived from melanomacell lines constituting both the initial (1990) and the modified (1993)human tumor cell line screens, are presented in Table 1 below. Alsoprovided in the table is information on the take-rate of the tumors, andthe experience of the NCI in the use of the tumors as early stage s.c.models. The doubling times were determined from vehicle-treated controlmice used in drug evaluation experiments (data for passage numbers 4-20are included). The doubling time is the median of the time interval forindividual tumors to increase in size from 200-400 mg (usually a periodof exponential growth). Both ranges and mean values are provided. Meandoubling times range from <2 d for some tumors (exemplified by theLOX-IMVI and SK-MEL-28 melanomas) to >10 d for the MALME-3M and M19-MELmelanomas.

[0181] The in vivo growth characteristics of the xenografts determinetheir suitability for use in the evaluation of test agent antitumoractivity, particularly when the xenografts are utilized as early stages.c. models. As used herein, an early stage s.c. model is defined as onein which tumors are staged to 63-200 mg prior to the initiation oftreatment. Growth characteristics considered in rating tumors includetake-rate, time to reach 200 mg, doubling time, and susceptibility tospontaneous regression. As can be noted, the faster-growing tumors tendto receive the higher ratings. TABLE 1 Growth Characteristics ofsc-Implanted Human Melanoma Xenografts In vitro panel Mean volumeUsefulness status doubling time Take as early-stage Line 1990 1993(range) in days Rate sc model LOX-IMVI Yes Yes 1.5 (1.1-2-1) Good GoodSK-MEL-28 Yes Yes 1.9 (1.1-2.5) Good Good UACC-62 Yes Yes 2.9 (1.9-4.2)70-80% Not Acceptable UACC-257 Yes Yes 5.4 (3.8-7.7) Good AcceptableSK-MEL-2 Yes Yes 5.7 (4.9-6-6) 80-90% Not Acceptable M14 Yes Yes 6.7(2.9-12.7) Good Acceptable SK-MEL-5 Yes Yes 7.3 (5.1-8-2) GoodAcceptable MALME-3M Yes Yes 11.2 (7.1-16.9) 90-90% Not AcceptableM19-MEL Yes No 12.3 (8.7-16.8) 60-90% Not Acceptable

Advanced-Stage Subcutaneous Xenograft Models

[0182] Such s.c.-implanted tumor xenograft models are used to evaluatethe antitumor activity of test agents under conditions that permitdetermination of clinically relevant parameters of activity, such aspartial and complete regression and duration of remission (Martin D S etal., Cancer Treat Rep 68:37-38 (1984); Martin D S et al., Cancer Res.46:2189-2192 (1986); Stolfi, R L et al., J. Natl Canc Inst 80:52-55(1988)). Tumor growth is monitored and test agent treatment is initiatedwhen tumors reach a weight range of 100-400 mg (staging day, medianweights approx. 200 mg), although depending on the xenograft, tumors maybe staged at larger sizes. Tumor sizes and body weights are obtainedapproximately 2 times/wk. Through software programs (developed by staffof the Information Technology Branch of DTP of the NCI), data arestored, various parameters of effects are calculated, and data arepresented in both graphic and tabular formats. Parameters of toxicityand antitumor activity are defined as follows:

[0183] 1. Toxicity: Both drug-related deaths (DRD) and maximum percentrelative mean net body weight losses are determined. A treated animal'sdeath is presumed to be treatment-related if the animal dies within 15 dof the last treatment, and either its tumor weight is less than thelethal burden in control mice, or its net body weight loss at death is20% greater than the mean net weight change of the controls at death orsacrifice. A DRD also may be designated by the investigator. The meannet body weight of each group of mice on each observation day iscompared to the mean net body weight on staging day. Any weight lossthat occurs is calculated as a percent of the staging day weight. Thesecalculations also are made for the control mice, since tumor growth ofsome xenografts has an adverse effect on body weight.

[0184] 2. Optimal % T/C: Changes in tumor weight (A weights) for eachtreated (T) and control (C) group are calculated for each day tumors aremeasured by subtracting the median tumor weight on the day of firsttreatment (staging day) from the median tumor weight on the specifiedobservation day. These values are used to calculate a percent T/C asfollows:

% T/C=(ΔT/ΔC)×100 where ΔT≧0 or =(Δi T/T_(I))×100 where ΔT<0  (1)

[0185] and T_(I) is the median tumor weight at the start of treatment.The optimum (minimum) value obtained after the end of the first courseof treatment is used to quantitate antitumor activity.

[0186] 3. Tumor growth delay: This is expressed as a percentage by whichthe treated group weight is delayed in attaining a specified number ofdoublings; (from its staging day weight) compared to controls using theformula:

[(T−C)/C]×100  (2)

[0187] where T and C are the median times (in days) for treated andcontrol groups, respectively, to attain the specified size (excludingtumor-free mice and DRDs). The growth delay is expressed as percentageof control to take into account the growth rate of the tumor since agrowth delay based on (T−C) alone varies in significance withdifferences in tumor growth rates.

[0188] 4. Net log cell kill: An estimate of the number of log₁₀ units ofcells killed at the end of treatment is calculated as:

{[(T−C)−duration of treatment]×0.301/median doubling time}  (3)

[0189] where the “doubling time” is the time required for tumors toincrease in size from 200 to 400 mg, 0.301 is the log₁₀ of 2, and T andC are the median times (in days) for treated and control tumors toachieve the specified number of doublings. If the duration of treatmentis 0, then it can be seen from the formulae for net log cell kill andpercent growth delay that log cell kill is proportional to percentgrowth delay. A log cell kill of 0 indicates that the cell population atthe end of treatment is the same as it was at the start of treatment. Alog cell kill of +6 indicates a 99.9999% reduction in the cellpopulation.

[0190] Tumor regression: The importance of tumor regression in animalmodels as an end point of clinical relevance has been propounded byseveral investigators (Martin et al., 1984, 1986 supra; Stolfi et al.,supra). Regressions are defined-as partial if the tumor weight decreasesto 50% or less of the, tumor weight at the start of treatment withoutdropping below 63 mg (5×5 mm tumor). Both complete regressions (CRs) andtumor free survivors are defined by instances in which the tumor burdenfalls below measurable limits (<63 mg) during the experimental period.The two parameters differ by the observation of either tumor regrowth(in CR animals) or no regrowth (=tumor-free) prior to the finalobservation day. Although one can measure smaller tumors, the accuracyof measuring a s.c. tumor smaller than 4×4 mm or 5×5 mm (32 and 63 mg,respectively) is questionable. Also, once a relatively large tumor hasregressed to 63 mg, the composition of the remaining mass may be onlyfibrous material/scar tissue. Measurement of tumor regrowth followingcessation of treatment provides a more reliable indication of whether ornot tumor cells survived treatment.

[0191] Most xenografts that grow s.c. may be used in an advanced-stagemodel, although for some tumors, the duration of the study may belimited by tumor necrosis. As mentioned previously, this model enablesthe measurement of clinically relevant parameters and provides a wealthof data on the effects of the test agent on tumor growth. Also, bystaging day, the investigator is ensured that angiogenesis has occurredin the area of the tumor, and staging enables “no-takes” to beeliminated from the experiment. However, the model can be costly interms of time and mice. For slower-growing tumors, the passage timerequired before sufficient mice can be implanted with tumors may be atleast ˜4 wks, and an additional 2-3 wks may be required before thetumors can be staged. To stage tumors, more mice (as many as 50-100%more) than are needed for actual drug testing must be implanted.

Early Treatment and Early Stage Subcutaneous Xenograft Models

[0192] These models are similar to the advanced-stage model, but,because treatment is initiated earlier in the development of the tumor,useful tumors are those with ≧90% take-rate (or <10% spontaneousregression rate). The “early treatment model” is defined as one in whichtreatment is initiated before tumors are measurable, i.e., <63 mg. The“early stage” model as one in which treatment is initiated when tumorsize ranges from 63-200 mg. The 63-mg size is used because it indicatesthat the original implant, about 30 mg, has demonstrated some growth.Parameters of toxicity are the same as those for the advanced-stagemodel; parameters of antitumor activity are similar. % T/C values arecalculated directly from the median tumor weights on each observationday instead of being measured as changes (Δ) in tumor weights, andgrowth delays are based on the days after implant required for thetumors to reach a specified size, e.g., 500 or 1000 mg. Tumor-free miceare recorded, but may be designated as “no-takes” or spontaneousregressions if the vehicle-treated control group contains >10% mice withsimilar growth characteristics. A “no-take” is a tumor that fails tobecome established and grow progressively. A spontaneous regression(graft failure) is a tumor that, after a period of growth, decreases to≦50% of its maximum size. Tumor regressions are not normally recorded,since they are not always a good indicator of antineoplastic effects inthe early stage model. A major advantage of the early treatment model isthe ability to use all implanted mice, which is why a good tumortake-rate is required. In practice, the tumors most suitable for thismodel tend to be the faster-growing ones.

Challenge Survival Models

[0193] In another approach, the effect of human tumor growth on thelifespan of the host is determined. The LOX-IMVI melanoma has been usedin this model. All mice dying or sacrificed owing to a moribund state orextensive ascites prior to the final observation day are used tocalculate median day of death for treated (T) and control (C) groups.These values are then used to calculate a percent increase in life span(“ILS”) as follows:

% ILS=[(T−C/C]×100  (4)

[0194] Where possible, titration groups are included to establish atumor doubling time for use in log₁₀ cell kill calculations. A death (orsacrifice) may be designated as drug-related based on visualobservations and/or the results of necropsy. Otherwise, treated animaldeaths are-designated as treatment-related if the day of death precedesthe mean day of death of the controls (−2SD) or if the animal dieswithout evidence of tumor within 15 days of the last treatment.

Response of Xenograft Models to Standard Agents

[0195] In obtaining drug sensitivity profiles for the advanced-stages.c. xenograft models, the test agent is evaluated following i.p.administration at multiple dose levels. The activity ratings are basedon the optimal effects attained with the maximally tolerated dose(<LD₂₀) of each drug for a given treatment schedule which is selected onthe basis of the doubling time of a given tumor, with longer intervalsbetween treatments for slower growing tumors.

[0196] The experience with melanomas described in Plowman, J. et al.,supra, is summarized in Table 2 below. At least minimal antitumoreffects (% T/C≧40) were produced in the melanoma group by at least 2,and as many as 10, clinical drugs. The number of responses appeared tobe independent of doubling time and histological type with a range inthe number of responses observed for tumors (seen in each subpanel ofother tumor types as well). When the responses are considered in termsof the more clinically relevant end points of partial or complete tumorregression, these tumors models (across all tumors) were quiterefractory to standard drug therapy; the tumors did not respond to anyof the drugs tested in 30 of 48 (62.5%) of all tumors. The melanomagroup shown was even more refractory to the standard drugs.

Strategy for Initial Compound Evaluation In Vivo

[0197] The in vitro primary screens provide a basis for selecting themost appropriate tumor lines to use for follow-up in vivo testing, witheach compound tested only against xenografts derived from cell linesdemonstrating the greatest sensitivity to the agent in vitro. The earlystrategy for in vivo testing emphasized the treatment of animals bearingadvanced-stage tumors. Examples of the in vivo data obtained with onesuch agent (Plowman, J. et al., supra) are summarized in Table 3. Aquinocarmycin derivative DX-52-1, identified as a melanoma-specificagent in vitro, demonstrated statistically significant antitumoractivity against 5/7 melanoma xenografts following ip administration onintermittent schedules (Plowman J et al., Cancer Res. 55:862-867 (1995).The most effective in vivo activity was observed against the rapidlydividing LOX-IMVI melanoma. TABLE 2 Response of Staged s.c. HumanMelanoma Xenografts to Clincal Anticancer Drugs Number of drugs activeTumor Minimal activity^(a) Tumor regression^(b) LOX-IMVI  7/10 0SK-MEL-28 2 0 UACC-62 9 0 SK-MEL-31 3/9 0/9 UACC-257 7 0 SK-MEL-2 7 0M14 3 0 MALME-3M 7 1

[0198] TABLE 3 Response of Advanced-Staged s.c. Human MelanomaXenografts to the Quinocarmycin Derivative, DX-52-1 Optimal Growth Dose(ip, % T/C delay: Regressions Melanoma Treatment days mg/kg/day)^(a)(Day)^(b) % (T-C)/C^(c) Complete-Partial LOX-IMVI 5,9,13 90 −54 181 2/10  3/10 5,9,13,17 60 −100^(b) 389 4/6 016 21,25 SK-MEL-2 14,21,28 9010 (32) 118 2/6 0/6 SK-MEL-5 15,19,23 40 49 (33)  27 0/6 0/6 UACC-6216,23,30 90 18 (34) 185 0/7 0/7 UACC-257 16,20,24 90 12 (27)  35 0/6 0/6M14 12,16,20 90 19 (26)  56 0/6 0/6 MALME-3M 27,31,35 90 64 (72)  4 0/60/6

[0199] Unless specific information is available to guide dose selection,single mice are preferably treated with single ip bolus doses of 100,200, and 400 mg/kg and observed for 14 d. Sequential 3dose studies maybe conducted as necessary until a nonlethal dose range is established.The test agent is then evaluated preferably in three s.c. xenograftmodels using tumors that are among the most sensitive to the test agentin vitro and that are suitable for use as early stage models. Thecompounds are administered ip, as suspensions if necessary, on schedulesbased, with some exceptions, on the mass doubling time of the tumor. Forexample, for doubling times of 1.3-2.5, 2.6-5.9, and 6-10 d, preferredschedules are: daily for five treatments (qd×5), every fourth day forthree treatments (q4d×3), and every seventh day for three treatments(q7d×3). For most tumors, the interval between individual treatmentsapproximates the doubling time of the tumors, and the treatment periodallows a 0.5-1.0 log₁₀ unit of control tumor growth. For tumors stagedat 100-200 mg, the tumor sizes of the controls at the end of treatmentshould range from 500-2000 mg, which allows sufficient time aftertreatment to evaluate the effects of the test agent before it becomesnecessary to sacrifice mice owing to tumor size.

Detailed Drug Studies

[0200] Once a compound has been identified as demonstrating in vivoefficacy in initial evaluations, more detailed studies are designed andconducted in human tumor xenograft models to explore further thecompound's therapeutic potential. By varying the concentration andexposure time of the tumor cells and the host to the drug, it ispossible to devise and recommend treatment strategies designed tooptimize antitumor activity.

[0201] The importance of “concentration×time” on the antitumor effectsof test agents were well illustrated by data obtained withamino-20M-camptothecin (Plowman, J. et al., 1997, supra). Those resultsindicated that maintaining the plasma concentration above a thresholdlevel for a prolonged period of time was required for optimaltherapeutic effects.

Hollow-Fiber Assays: A Newer Approach to In Vivo Drug Testing

[0202] This model uses human tumor cell lines growing in hollow fibersand is intended as a prioritization tool through which lead compoundsidentified in an in vitro screen would pass. In brief, tumor cells areinoculated into hollow fibers (1 mm internal diameter), and the fibersare heat-sealed and cut at 2-cm intervals. These samples are maintainedfor 24-48 h in vitro and then implanted into nude mice. At the time ofimplantation, a representative set of fibers is assayed for viable cellmass by the “stable end point” MTT dye conversion technique (Alley, M Cet al., Canc Res 51:1247-1256 (1991)) in order to determine the “timezero” cell mass for each cell line. The mice are treated with testagents on a daily treatment schedule, and the fibers are collected 6-8 dpostimplantation. At collection, the quantity of viable cells containedin the fibers is measured. The antitumor effects of the test agents aredetermined from the changes in viable cell mass in the fibers collectedfrom compound-treated and diluent-treated mice. Using this technique,three different tumor cell lines can be grown conveniently in each oftwo physiologic sites (e.g., ip and sc) within each experimental mouse.Thus, this model provides a method for administering a test agent ip toevaluate its effect against tumor cells growing in both the ip cavityand the s.c. compartment. Such simultaneous assessment of multiple tumorcell lines grown in two physiologic compartments should permit rapididentification of lead compounds with the greatest promise of clinicaleffectiveness.

[0203] This in vivo/in vitro hollow-fiber system may be well suited forthe prioritization of compounds for more advanced stages of in vivo drugevaluation. Practically, this system can be viewed as a means tofacilitate traditional chemotherapeutic testing, since it is rapid,sensitive, and is broadly applicable to a variety of human tumor celltypes. Additionally, it requires only a limited quantity of testcompound, a relatively small number of animals and, therefore, limitedanimal housing space.

Xenograft Model of Metastasis

[0204] The compounds of this invention are also tested for inhibition oflate metastasis using an experimental metastasis model such as thatdescribed by Crowley, C. W. et al., Proc. Natl. Acad. Sci. USA 905021-5025 (1993)). Late metastasis involves the steps of attachment andextravasation of tumor cells, local invasion, seeding, proliferation andangiogenesis. Human melanoma cells transfected with a reporter gene,preferably the green fluorescent protein (GFP) gene, but as analternative with a gene encoding the enzymes chloramphenicolacetyl-transferase (CAT), luciferase or LacZ, are inoculated into nudemice. This permits utilization of either of these markers (fluorescencedetection of GFP or histochemical calorimetric detection of enzymaticactivity) to follow the fate of these cells. Cells are injected,preferably iv, and metastases identified after about 14 days,particularly in the lungs but also in regional lymph nodes, femurs andbrain. This mimics the organ tropism of naturally occurring metastasesin human melanoma. For example, GFP-expressing melanoma cells (10⁶ cellsper mouse) are injected i.v. into the tail veins of nude mice. Animalsare treated with a test composition at 100 μg/animal/day given q.d. IP.Single metastatic cells and foci are visualized and quantitated byfluorescence microscopy or light microscopic histochemistry or bygrinding the tissue and quantitative calorimetric assay of thedetectable label.

A Human Melanoma/SCID Mouse Model

[0205] Safrians, S. et al., Int'l J. Canc. 66:131-1f58 (1996),incorporated by reference) described studies in a human melanoma/SCIDmouse model. The highly metastatic human melanoma line. C8161 (Welch etal., 1991) was transfected with antibiotic-selectable markers (with thevectors pSV2neo and pSV2hygro) using conventional methods. As clonesemerged when the cells were grown in medium containing G-418 andhygromycin, the concentrations of the two agents were reducedrespectively to 0.2 mg/ml and 0.1 mg/ml. Emerging clones were identifiedwithin 3-4 weeks and removed with cloning rings. Ploidy studies andkaryotype analyses were performed to verify that selected clones bearingeither of the two markers had no gross alterations in DNA content norhad they undergone changes in doubling time, tumorigenicity,constitutive levels of secreted collagenases, in vitro, Matrigelinvasion or, most importantly, metastatic phenotype. Both neo⁻ C8161 andhyg⁻ C8161, like the parental line, demonstrate strong cytoplasmicimmunoreactivity of cytokeratins 8 and 18, which facilitates theirdetection within the organs.

[0206] Between 5×10⁴ and 5×10⁶ neo⁻ and/or hyg⁻ C8161 cells suspended in0.2 ml Hanks' balanced salt solution (HBSS) are injected either s.c. ina right dorsolateral flank region (assay for spontaneous metastasis) ori.v. in the tail vein (hematogenous( ) metastasis) or via both routes atsuccessive intervals. Animals are killed at various intervals preferablyranging from 2 to 8 weeks), the organs are removed and metastaticcolonies are quantified to determine the distribution of tumor cellsfrom hematogenous dissemination. The size of the primary tumor as wellas the number and distribution of metastases are determined.

[0207] Representative mice are subjected to histopathological andimmunocytochemical studies to further document the presence ofmetastases throughout the major organs. Number and size (greatestdiameter) of the colonies can be tabulated by digital image analysis,e.g., as described by Fu, Y. S. et al., Anat. Quant. Cytol. Histol.11:187-195 (1989)).

[0208] For determination of colonies, explants of lung, liver, spleen,para-aortic lymph nodes, kidney, adrenal glands and s.c. tissues arewashed, minced into pieces of 1-2 mm³ and the pieces pulverized in aTekman tissue pounder for 5 min. The pulverized contents are filteredthrough a sieve, incubated in a dissociation medium (MEM supplementedwith 10% FCS, 200 U/ml of collagenase type I and 100 μg/ml of DNase typeI) for 8 hr at 37° C. with gentle agitation. Thereafter, the resultingcell suspension is washed and resuspended in regular medium (e.g., MEMwith 10% FCS supplemented with the selecting antibiotic (G-418 orhygromycin). The explants are fed as described by Safrians et al.,supra, and the number of clonal outgrowths of tumor cells is determinedafter fixation with ethanol and staining with a monoclonal antibody tocytokeratins 8 and 18. The number of colonies is counted over an 80-cm²area. If desired, a parallel set of experiments can be conducted whereinclonal outgrowths are not fixed and stained but rather are retrievedfresh with cloning rings and pooled after only a few divisions for othermeasurements such as secretion of collagenases (by substrate gelelectrophoresis) and Matrigel invasion.

[0209] Modified Matrigel invasion assays are performed as described byothers (Hendrix, M. J. C. et al., Cancer Lett., 38:137-147 (1987);Albini, A. et al., Cancer Res., 47 3239-3245 (1987); Melchiori, A.,Cancer Res. 52:2353-2356 (1992)). Substrate gel electrophoresis ofconditioned media from the aforementioned clones are analyzed asdescribed by others (Herron, G. S. et al., J. Biol. Chem. 261:2814-2818(1986); Ballin, M. et al., Biochem. Biophys. Res. Comm., 154:832-838(1988)).

[0210] All experiments are performed with groups that preferably have 10mice. Results are analyzed with standard statistical tests. C8161 cellsdemonstrate significant numbers of both spontaneous and hematogenousmetastasis. Significant numbers of hematogenous metastases may beproduced almost exclusively in the lungs with an injection of 5×10⁵ cell(and larger numbers result in extrapulmonary metastases).

[0211] Safrians et al., supra, found that i.v. injections of 5×10⁵ tumorcells 1 week after an s.c. flank injection of an equal number of tumorcells followed by an additional 5-week interval yielded a ratio of 2:1hematogenous:spontaneous pulmonary metastases and an overall pulmonarytumor burden of 1.25 g (over a normal pulmonary weight: 0.2 g). Withthis regimen, numerous extrapulmonary metastatic clones could beretrieved from spleen, liver, kidneys, adrenal gland, para-aortic lymphnodes and s.c. sites. The vast majority of these clones representspontaneous metastases from the locally growing tumor. Similar resultswere obtained with C8161 carrying either of the antibiotic resistancemarkers discussed above.

[0212] 3. Transgenic Mouse Model

[0213] A useful murine melanoma model in which dominantly actingoncoproteins are somatically regulated in vivo was developed by Chin, L.et al., Nature 400:468-472 (1999 July). Cohorts of single and doubletransgenic mice (designated Tyr/Tet-Ras) homozygous null for the INK4agene (INK4a^(−/−)) were generated.

Production of the Transgenic Mice

[0214] The reverse tetracycline transactivator (rtTA) is a 1,050 bpEcoRI/BamHI fragment isolated from pUHD172-1neo (Gossen, M. et al.,Science 268:1766-1769 (1995)). The Tet promoter contains an XhoI/EcoRIfragment of the cytomegalovirus minimal promoter linked to the tetoperator sequence. The tyrosinase enhancer/promoter and theH-Ras^(Va112) transgene were as described in Fasano, O. et al., J. Mol.Appl. Genet. 2:173-180 (1983); Chin, L. et al., Genes Dev. 11:2822-2834(1997); and Ganss, R. et al., EMBO J. 13:3083-3093 (1994). Fasano, O. etal., J. Mol. Appl. Genet. 2:173-180 (1983). Chin, L. et al. (Nature400:468-472 (1999)) generated multiple founder lines for both transgenesand used one activator line (Tyr/rtTA, line 37) and two independentreporter lines (Tet-RAS, lines 65 and 72) for further development.

Primary Tumors, Derivative Cell Lines and SCID Explant Tumors

[0215] Transgenic mice are fed doxycycline drinking water (2 mg/ml insucrose water) and observed for spontaneous tumor development. Primarytumors are adapted to culture by mechanical mincing with sterilizedrazor blades and brief trypsinization, and maintained in RPMI mediumcontaining 10% serum and supplemented with doxycycline (2 μg/ml medium)(Kistner, A. et al., Proc. Natl. Acad. Sci USA 93:10933-10938 (1996)).

[0216] For the SCID explant tumors, 2-5×10⁶ established melanoma cellare injected s.c. into the flanks of adult SCID mice maintained ondoxycycline or regular drinking water. These cell lines are passagedsufficiently to insure elimination of immunocytes from the originalhost.

Development of Melanomas after Doxycycline Treatment

[0217] As indicated above, double transgenic mice are generated byintercrossing INK4a^(+/−) mice with either

[0218] (a) a transgenic mouse line harboring the rtTA under the controlof the tyrosinase gene promoter/enhancer elements (designated Tyr-rtTA),or

[0219] (b) a transgenic mouse line containing the H-Ras^(V12G) openreading frame driven by a minimal promoter containing multimerizedtet-operons, designated Tet-Ras (Gossen et al., supra; Fasano et al.,supra).

[0220] In the doxycycline-treated group, about 25% of double transgenicTyr/Tet-Ras INK4a^(−/−) mice develop melanomas (average latency: 60days). In contrast, untreated Tyr/Tet-Ras INK4a^(−/−) mice or treatedsingle transgenic Tet-Ras INK4a^(−/−) mice do not develop melanomas.

[0221] The Tyr/Tet-Ras INK4a^(−/−) animals' melanomas shared all of themacroscopic features of the melanomas of constitutive Tyr-RasINK4a^(−/−) mice, manifesting as amelanotic, invasive and highlyvascular tumors, reminiscent of nodular-type melanoma in humans.Characteristics include spindle morphology with anaplastic andpleiomorphic cytology, and strong immunoreactivity to the earlymelanocyte-specific marker tyrosinase-related-protein-I (TRP-1)(Thomson, T. M. et al., J. Invest. Dermatol. 90:459-466 (1988)). Tumorsand cultured cell lines derived therefrom express strong H-Ras^(V12G)expression and activity.

[0222] These double transgenic animals that can be induced to developmelanoma are treated in accordance with the present invention toevaluate the anti-melanoma activity of the agents described herein.

[0223] For a compound to be useful in accordance with this invention, itshould demonstrate activity in at least one of the in vitro or in vivoassay systems described herein.

[0224] Having now generally described the invention, the same will bemore readily understood through reference to the following exampleswhich are provided by way of illustration, and are not intended to belimiting of the present invention, unless specified.

EXAMPLE I Materials and Methods for Studies of Melanoma Cells CellLines, Reagents and Treatments

[0225] Human melanoma cell lines used in this study were obtained fromNCI-ADS and cultured as described (Monks, A. et al. J Natl Canc Inst83:757-766 (1991)). Normal neonatal human epidermal melanocytes (NHEM2489) were purchased from Clonetics and cultured in Melanocyte GrowthMedium-3 (Clonetics) as suggested by the manufacturer. Purifiedrecombinant protective antigen (PA), lethal factor (LF) and edema factor(EF) were provided by Stephen H. Leppla (National Institute of DentalResearch, NIH, USA) and were used to treat cells at 0.1 μg/ml each inthe appropriate culture medium. Control groups were treated with PAalone, which functions as a translocator for LF and EF (Leppla, S. H.,supra; Duesbery, N. S. et al., 1999, supra). When treating with LF orEF, their respective toxin complexes that included PA were used (“LF+PA”and “EF+PA”). For normal melanocytes, additional LF+PA or PA alone (0.1μg/ml each) was added directly to the medium 72 hours after the initialtreatment, and the cells were incubated for an additional 24 hours.

[0226] PD98059 (New England Biolabs, Inc.) was dissolved in dimethylsulfoxide (DMSO), and 20 mM aliquots were stored at −20° C. To maintainsustained inhibition of MEKs, PD98059 was directly added to the culturesevery 24 hours to achieve a final concentration of 20 μM for theduration of the study, unless otherwise indicated. IBMX (Calbiochem Co.)dissolved in DMSO was added to the appropriate culture medium at a finalconcentration of 200 μM. An equivalent volume of DMSO solvent was addedto controls. U0126 (Promega Corp) was dissolved in DMSO to 10 mM, and asingle dose, final concentration of 10 μM, was used.

Quantitation of Apoptosis

[0227] Apoptosis was quantified as described previously (Koo, H. -M. etal. J Natl Canc Inst 91:236-244 (1999); Koo, H. -M. et al. Canc Res59:6057-6062 (1999)). Briefly, total cells including floating cells werecollected, washed in phosphate-buffered saline (PBS), and fixed in 10%formalin. The fixed cells were washed in PBS, and then mounted onto apolylysine-coated glass slide by Cytospin centrifugation. The mountedcells were stained for DNA with 4′6-diamidine-2′-phenylinedihydrochloride (DAPI) and examined by fluorescent microscopy. A totalof 300-500 nuclei from several random fields were examined, andapoptosis was expressed as a percentage of the nuclei displayingapoptosis-associated morphological changes.

Flow Cytometry

[0228] Cell cycle profiles and DNA content were analyzed by flowcytometry, as previously described (Koo, H. -M. et al. Canc Res59:6057-6062 (1999)). Total cells including floating cells wereharvested, washed in cold PBS and fixed in cold 70% ethanol overnight.The fixed cells were rehydrated in PBS containing 1% fetal bovine serum,washed in PBS, incubated with DNase-free RNase at room temperature for30 min, and then stained with 50 μg/ml propidium iodide overnight at 4°C. in the dark. The stained samples were analyzed in a FACSCalibur(Becton Dickinson Biosciences) for DNA content and cell cycle profiles.

Western Immunoblotting

[0229] Western blot analysis was performed as described previously (Kooet al., 1999, supra). The primary antibodies used were anti-active MAPKfrom Promega, and anti-ERK1 (K-23) and anti-ERK2 (C-14) from Santa CruzBiotechnology.

Determination of Melanin Content

[0230] Intracellular melanin was solubilized by lysing cells in 0.2NNaOH and incubating at 60° C. m for 1 hour. The relative absorbance at405 nm for melanin to that at 280 nm for protein was calculated(A₄₀₅/A₂₈₀). The control relative absorbance, “A₄₀₅/A₂₈₀ (PA alone)”wasset to 1.0.The melanin content in each treatment group was expressed asthe ratio

[0231]A₄₀₅/A₂₈₀ (experimental)

[0232] A₄₀₅/A₂₈₀ (PA alone)

[0233] To visualize melanization, 4×10⁶ cells from each group wereconcentrated in a well of 96-well microplate and photographed (e.g.,FIG. 5).

In vivo Testing of LF on M14-MEL or SK-MEL-28 Xenografts in Athymic NudeMice

[0234] Female athymic nude (nu/nu) mice (˜8 weeks old) were injecteds.c. with M14-MEL or SK-MEL-28 cells (10⁷ cells/mouse). When tumors grewto an average mass of ˜95 mg in the earlier experiment or about 450mm³for M14-MEL and about 310 mm³ for SK-MEL-28 in the latter experiments,the control group (n=6) was treated with PA (6 μg/mouse) alone by dailyintratumoral injection for 13 days. During the same period, the testgroup (n=7) was treated first with 6 μg PA+2 μg LF/mouse for 4 days andthen continued with 6 μg PA+4 μg LF/mouse for another 9 days.

[0235] M14-MEL xenograft tumors (n=6) were treated with 7 doses, andSK-MEL-28 tumors (n=7) with 5 doses and then each animal was given anadditional dose three days later. Tumor weight was calculated by theformula: (L×W²)/2. The experiment was terminated when a majority of micein the control group showed a significant weight loss (>10% of bodyweight) or when control tumors reached about 2000 mm³.

Histology and in Situ TUNEL Staining

[0236] Dissected tumors were fixed in 10% neutral-buffered formalinovernight, embedded in paraffin blocks, cut into thin (5 μm-thick)sections and then mounted on glass slides. After deparaffinization andrehydration, tumor sections were stained with hematoxylin and eosin(H&E) or for TUNEL. TUNEL staining was performed using “In Situ CellDeath Detection, POD kit (Roche Molecular Biochemicals) as recommendedby the manufacturer. Tumor sections were counter-stained withhematoxylin. TUNEL-positive areas stain dark brown and nuclei (or DNA)stain light-blue

EXAMPLE II

[0237] MEK-Directed Protease LF and PD98059 Induce Apoptosis inDifferent Human Melanoma Cell Lines

[0238] Following 72-hour incubation, all melanoma cell lines testedunderwent a variable degree of apoptosis in response to LF (FIG. 1). Thesame cohort of melanoma cell lines was also treated with PD98059. Forthis experiment, the cells were repeatedly exposed to the inhibitorevery 24 hours for 72 hours, because of instability of the compound inculture medium Dudley, D. T. et al., Proc Nat'l Acad Sci USA92:7686-7689 (1995); Alessi, D. R. et al., J Biol Chem 270:27489-27494(1995)) Again, all of the cell lines responded to the MEK inhibitor byapoptosis (FIG. 2).

[0239] With the exception of the LOX-IMVI cells, which express differentgenes from melanoma cells (Stinson, S. F. et al., Anticancer12:1035-1054 (1992); Ross, D. T. et al., Nature Genetics 24:227-235(2000)) the relative sensitivities of the melanoma lines to apoptosisinduced by LF and PD98059 were similar (cf. FIG. 1 and FIG. 2).

[0240] Apoptosis could mostly account for the enhanced sensitivity ofhuman melanoma cell lines in the NCI-ADS to LF or PD98059.

[0241] Another MEK inhibitor, U0126, also induced apoptosis in thesemelanoma lines with a similar pattern (FIG. 15).

[0242] These results demonstrate that human melanoma cells areespecially sensitive to apoptosis triggered by inhibition of MAPKsignaling. This is unlike most other cell types so far tested, which, inresponse to the MAPK signal blockade, display cytostatic growthinhibition without cell death both in vitro and in vivo (Cohen, P. CurrOpin Chem Biol 3:459-465 (1999); Roussel, M. F., Adv Canc Res, 74:1-241998); Sebolt-Lepold, J. S. et al., supra.

EXAMPLE III Inhibition of MAPK Activation Affects Melanoma Cell CycleProgression

[0243] In most cell types, activation of the MAPK pathway is requiredfor progression through the restriction point in G1 phase of the cellcycle (reviewed in Roussel et al., supra). To investigate the effect ofinhibition of MAPK activation on melanoma cell cycle progression, cellcycle changes and apoptosis were monitored in a representative melanomacell line (MALME-3M) during a 72-hour exposure to LF or PD98059.

[0244] Within 24 hours of the treatment with LF or PD98059, cell cycleprogression was noticeably blocked in the G1 phase (FIGS. 3B and 3E).Both agents induced apoptosis by 48 hours, and a significant fraction ofthe cells (>25%) were undergoing apoptosis by 72 hours (FIGS. 3C, 3D, 3Fand 3G).

[0245] Continuous accumulation of the cells in G1 phase was noted asevidenced by a decrease in the percentage of cells in the S and G2/Mphases (FIG. 3A-3G). In both LF and PD98059-treated cells, activation ofERK1 and 2 was inhibited (FIG. 4).

[0246] After a 72-hour incubation with LF or PD98059, a similar responseof apoptosis and G1 arrest, coinciding with down-regulation of ERKactivation was observed in all the other human melanoma cell linesexamined (see Table 4). Nevertheless, these responses were reversiblesince, even after 72-hours with LF or PD98059, removal of the inhibitorsallowed restoration of ERK activation and resumption of normal cellcycle progression.

[0247] It was concluded that sustained inhibition of the MAPK signalingpathway is required to effectively trigger apoptosis in human melanomacells.

EXAMPLE IV Induced Melanoma Cell Differentiation and Apoptosis

[0248] Both LF and PD98059 were found to induce melanin production inthe melanotic melanoma cell lines tested. Melanogenesis is inducedduring melanocyte (and melanoma cell) differentiation (Busca, R. et al.Mol Biol Cell 9:1367-1378 (1998)). Inhibition of the MAPK pathway wasknown to induce differentiation of B16 mouse melanoma cells (Englaro, W.et al. J Biol Chem 273:9966-9970 (1998)). These findings suggested thatapoptosis may be associated with terminal differentiation induced byinhibition of the MAPK pathway. To address this possibility, cyclic AMP(cAMP)-elevating agents that induce melanoma cell differentiation (Buscaet al., supra, and reference cited therein) were examined for theireffects on melanogenesis and apoptosis induced by LF or PD98059 in themelanotic UACC-257 and MALME-3M cell lines.

[0249] The cAMP-elevating agents used were the edema factor (EF) ofBacillus anthracis, an adenylyl cyclase (Leppla, supra; Duesbery et al.,supra), and isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor(Beavo, J. et al., Trends Pharmacol Sci 11:150-155 (1990)).

[0250] Neither EF nor IBMX treatment induced significant melanogenesisin the human melanoma cell lines examined. However, combination of EF orIBMX with LF or PD98059 caused a synergistic effect on the melaninproduction (FIGS. 5 and 6). Morphological changes (dendrite formation)induced by LF or PD98059 was also enhanced by EF or IBMX in bothmelanoma lines.

[0251] The cooperation between LF and EF on melanization may explain theformation of a blackened eschar in the skin lesion of anthrax infectionsite (Leppla, supra; Duesbery et al., supra). See the photographs(circles) in FIG. 5A.

[0252] In contrast, apoptosis induced by LF or PD98059 was severelyantagonized by simultaneous treatment with EF or IBMX (FIGS. 6 and 8).This antagonism occurred even though activation of ERK1 and 2 wassignificantly inhibited (FIGS. 7 and 9).

[0253] The partial inhibition (or restoration) of activation of ERK1/2by PD98059 was sufficient to trigger apoptosis in MALME-3M cells,whereas the greater degree of inhibition produced by the combination ofPD98059+IBMX was not apoptotic (FIG. 9).

[0254] A similar antagonistic effect of EF on LF-induced apoptosis wasobserved in all the other human melanoma cell lines tested.

[0255] These results demonstrated that apoptosis in melanoma cells wasnot merely a “byproduct” of the differentiation induced by inhibitingMAPK signaling. These results suggested that (1) an event or eventsdownstream from MAPKs is or are responsible for the apoptotic responseof γmelanoma cells and (2) this event(s) is dominantly modulated by acAMP-dependent pathway. TABLE 4 LF and Low Molecular Weight MEKInhibitors PD98059 and U0126 Induced Apoptosis in Human Melanoma CellLines. Experiment I Experiment II Apoptosis SD (%) Apoptosis (%)Melanoma cell lines LF PD98059 U0126 LF LF + EF LOX-IMVI* 49.2 4.5 3.61.8 2.3 1.8 47.2 20.8 MALME-3M 55.2 2.6 47.9 3.3 48.3 10.3 31.5 1.9M14-MEL 51.9 0.9 53.0 2.7 36.0 8.7 37.1 42.4 SK-MEL-2 14.9 3.0 23.4 2.335.4 4.4 19.1 8.5 SK-MEL-28 8.2 1.4 2.5 0.6 1.1 0.2 8.4 0 SK-MEL-5 6.70.6 9.1 1.2 1.9 0.5 3.7 1.5 UACC-257 8.6 1.1 19.7 4.0 4.7 1.2 32.1 1.0UACC-62 28.1 1.2 30.5 1.8 8.3 0.9 20.4 17.3

[0256] The results with a different small molecule inhibitor of MEKinhibitor, PD184352 (Sebolt-Leopold et al., supra) are shown in Table 5,below. TABLE 5 Dose-Dependent Apoptotic Response of Human M14-MELMelanoma Cells to PD184352 Treatment for 72 hours % Apoptosis M14-MELMelanoma Cells DMSO 0.6 PD98059 20 μM 38.9 PD184352  1 μM 24.5 PD184352 5 μM 37.3 PD184352 10 μM 48.8 PD184352 20 μM 71.0 Normal MelanocytesDMSO 0 PD98059 20 μM 0 PD184352  2 μM 0.3

[0257] The above results show that PD184352 is selectively cytotoxic tohuman melanoma cells in comparison to normal human melanocytes and thatthis cytotoxic action occurs via apoptosis. Indeed this compound acts ina dose-dependent manner and appears to be more potent, possibly byseveral fold, than PD98059

[0258] Western blot analysis with a phospho-specific anti-ERK antibodyconfirmed that activation of MAPK (ERK1/2) was completely blocked byPD184352 in M14-MEL melanoma cells.

EXAMPLE V Inhibition of MAPK Signaling in Normal Human Melanocytes

[0259] Incubation of normal melanocytes for 72 hours with PD98059 (andU0126) or 96 hours with LF induced G₁ arrest, and while MAPK activationwas inhibited, no apoptosis was observed (FIG. 12B and 13).

[0260] A 96-hour incubation with LF completely inhibited ERK activation(FIG. 9), but no apoptosis was detected (FIG. 11B). In contrast, ashorter (72 hours) incubation with LF triggered apoptosis in arepresentative melanoma cell line (FIG. 11D). Similarly, apoptosis wasnot detected in normal melanocytes treated with PD98059 for 96 hours(FIG. 11B). These results indicate that the MAPK cascade generates ormediates a survival signal that is specific to malignant melanoma cellsand that is not essential for survival of normal melanocytes in culture.

[0261] This is consistent with the findings of Chin, L. et al (Nature400:468-472 (1999)) that in a mouse melanoma model null for INK4a andcontaining a doxycycline (Dox)-inducible ras oncogene, withdrawal of Dox(thus ras oncogene down-regulation) results in regression by markedapoptosis of primary and explanted tumor cells. The rasoncogene-dependency of melanoma cell maintenance in this model could beexplained by the existence of a melanoma cell-specific survival pathwaypositively regulated by the MAPK cascade that can be constitutivelyactivated by ras oncogene activation or upstream receptor tyrosinekinase activation Lewis, T. S. et al., Adv Canc Res 74:49-139 (1998);Roussel et al., supra).

EXAMPLE VI Anthrax Lethal Factor and PD98059 Act on Various other Tumors

[0262] Unexpectedly, among the breast carcinoma cell lines in NCI-ADS,two related cell lines MDA-MB-435 and MDA-N were as sensitive asmelanoma cell lines to the growth inhibition rendered by LF or PD98059.(See Table 5.) However, recent cDNA microarray analysis of approximately8,000 different genes revealed that these cell lines were derived from asingle patient with breast cancer (Cailleau, R. et al., In Vitro14:911-915 (1978)) and exhibited the gene expression profile morecharacteristic of melanoma cells, suggesting that these cells may haveoriginated from a melanoma rather than the mammary adenocarcinoma (Rosset al., supra).

[0263] These findings not only provide an explanation for the enhancedsensitivity of these tumor cells to LF or PD98059, but also corroboratethe present inventors' finding that human melanoma cells arespecifically sensitive to inhibition of the MAPK pathway.

[0264] The incidence of malignant melanoma is increasing dramatically inall parts of the world (Parker, S. et al., CA Cancer J Clin 47:5-27(1997)). Despite improved diagnosis and characterization, long-termsurvival from the advanced disease has not improved. The presentfindings that inhibition of MAPK signaling specifically triggers anapoptotic response in human melanoma cells, but not in normalmelanocytes or in other cell types, makes this a novel selectivetherapeutic strategy for systematic treatment of malignant melanomas.TABLE 5 Sensitivity of Human Tumor Cell Lines of NCI-ADS Cancer Panelsto LF and PD98059* Treatment Melanoma panel Other tumor panel † LF 0.63ng/ml 540 ng/ml PD98059 1.61 × 10⁻⁵ M 5.04 × 10⁻⁵ M

EXAMPLE VII MAPK Pathway Inhibition In Vivo Inhibits Tumor Growth

[0265] To evaluate the effects of MAPK pathway inhibition on growth ofmelanoma in vivo, M14-MEL or SK-MEL-28 cells were xenografted s.c. intofemale athymic nude mice (NCR nu/nu mice about 8 weeks old). Results areshown in FIGS. 14-18. When the tumors became established (mass of ˜95mg), treatment with LF was initiated (Day 13). The test group (n=7) wastreated daily first with 2 μg of LF (+6 μg of PA)/mouse for 4 days (Day13-16) and then with 4 μg of LF (+6 μg PA)/mouse for another 9 days (Day17-25). The control group (n=6) was treated with PA alone (6 μg/mouse)daily during the same period (Day 13-25).

[0266] Treatment with LF significantly attenuated the growth of M14-MELtumors in vivo. Furthermore, a dose effect was observed: growthinhibition was greater with 4 μg of LF than with 2 μg of LF and thetumors grew at a faster rate when treatment was stopped. Tumor growthwas not affected by treatment with PA alone. Tumor size varied more inanimals treated with PA alone than in those treated with LF. Theinhibitory effects on tumors were also manifest as a difference in tumordepth.

[0267] Histological examination (not shown) of the tumors furtherrevealed qualitative differences of LF-treatment compared to the control(PA alone). Tumors in PA-treated subjects showed signs of hemorrhage,while tumors treated with LF were occupied with a central white mass. Toexamine cellular structure and apoptosis, tumor sections were stainedwith H&E and by TUNEL, respectively. Cellular organization was absent inthe white mass in tumors of the LF-treated group and these exhibitedstrong TUNEL positivity, especially at the junction between the whitemass and light-brown layer. Higher magnification revealed that nucleiwere severely fragmented, a typical sign of apoptosis. Thus, it wasconcluded that after LF treatment, tumors were filled with dead andapoptotic cells (white mass), and this region was surrounded by livetumor cells (light-brown layer). In PA-treated tumors, the hemorrhagiczone scored positive for TUNEL because of the endogenous peroxidaseactivity of the red blood cells. Importantly, TUNEL-positive apoptoticcells were not seen.

[0268] LF prevented M14-MEL tumors from growing (40% overall reductionin tumor size at day 31, FIG. 16A). When the LF treatment was halted forM14-MEL tumors, one tumor continued to regress, while the majorityslowly resumed growth (FIG. 16A). LF had a much more dramatic effect onSK-MEL-28 xenografts (FIG. 16B). All tumors in this series regressedcompletely during LF treatment, and the animals remained tumor-free forup to 5 weeks (FIG. 16B). PA alone had no effect on tumor growth ineither study. Unexpectedly, SK-MEL-28 melanoma cells were more sensitiveto LF in vivo than in vitro (Table 4) suggesting a stronger requirementof the MEK/MAPK signaling for melanoma cell survival in vivo. Thevariation in response of M14-MEL and SK-MEL-28 tumors to LF may be dueto the differences in in vivo growth rate (FIG. 16) and in tumor size atinitial treatment. Histological characterization revealed qualitativedifferences between the LF- and PA-treated tumors.

[0269]FIG. 18 also shows the effect of LF treatment of SK-MEL-28 bydirect intratumoral injection. When tumor growth was established (volumeof ˜310 mm³ on day 27 after implantation), tumors were treated with 6 μgPA+2 μg LF/mouse (PA+LF) or 6 μg PA/mouse as controls). Five doses weregiven at 2-day intervals over days 27-35 and then an additional dose wasgiven on day 38. The PA+LF-treated tumors remained in completeregression for at least 71 days. PA alone had no effect on tumor growth.Standard deviations (SD) of PA-treated tumors, ranged from 48 mm³ (day27) to 250 mm³ (day52); SD's of PA+LF-treated tumors ranged from 56 mm³(day 27) to 32 mm³ (day35).

[0270] Histological characterization revealed qualitative differencesbetween the LF- and PA-treated tumors. The following description is of acomparison of the effects of LF on two melanomas (see also discussionabove for M14-MEL results). Tumors of the control groups treated with PAalone showed hemorrhage and necrosis (FIG. 17A, 17B, and 17J, 17K). Thearea around necrotic foci (FIG. 17C) and sporadic tumor cells (FIG. 17L)stained positive for TUNEL. In contrast, the LF-treated M14-MEL tumorswere largely void of organized cellular structures and were packed withfragmented nuclei, which stained intensely for TUNEL (FIG. 17D-17I),indicating apoptotic cell death. Some surviving tumor cells, however,were detected at the periphery of the M14-MEL tumor (FIG. 17D-17F). InSK-MEL-28 tumors, all tumor cells remaining were strongly stained (FIG.17M-17R). Moreover, melanin deposits were evident throughout theLF-treated tumors (insets in FIGS. 17E, 17H, 17N and 17Q), consistentinduction of melanin production by LF. LF had no apparent side effectsduring and following treatment.

[0271] These results demonstrated that the strategy of inhibiting theMAPK pathway triggered cell death by apoptosis in melanomas growing invivo.

[0272] Results in FIG. 19 show an inverse relationship between the tumorsize and the antitumor effects of LF treatment. The PA+LF treatmentregimen caused complete regression of the three groups of tumors thatwere initially smaller (76, 187, and 278-mm³) and approximately 40%overall reduction in larger tumors (initial size—443-mm³). PA alone hadno effect on tumor growth. At the end of the treatment interval (day14), “control” tumors treated with PA showed significant variation involume (SD from 109 to 263 mm³), whereas, only tumors of the 443-mm³group survived PA+LF treatment varied in volumes (SD from 23 to 131mm³).

EXAMPLE VIII LF Administered Intravenously Inhibits Growth of MALME-3MMelanoma Xenograft

[0273] Three nude mice bearing MALME-3M tumors were injected IV in thelateral tail vein first with 2 μg LF/mouse followed by 20 μg PA/mouseone hour later. Results are shown in FIG. 21 Treatment was either daily(◯and □) or at 2-day intervals (♦). For the daily treatment animal, atotal of 4 doses was given; for the 2-day interval schedule, a total of7 doses were given (arrowheads). Both these systemic treatment protocolsresulted in tumor regression.

[0274] The IV treatment regimen selected for further studies was: IVinjection first with 2 μg LF/mouse and followed one hour later by 20 μgPA/mouse; this combination treatment was administered using 2-dayintervals. Based on the results in FIG. 21, the following regimen isused and the following outcomes are expected:

[0275] Tumors: Subcutaneous MALME-3M xenograft tumors in onedorsolateral flank (˜100 mm³). Treatment Protocol: Intravenous injection(tail vein) of 50 μl volumes TABLE 6 Treatment schedule at 2-dayintervals First IV Second IV Expected Group n injection Intervalinjection Results A ≧5 — — 20 μg PA/mouse Tumor Growth B ≧5  2 μg 15 min20 μg PA/mouse Regression LF/mouse C ≧5  2 μg 1 hour 20 μg PA/mouseRegression LF/mouse D ≧5 10 μg 1 hour 20 μg PA/mouse Regression LF/mouse

EXAMPLE IX Systemic Effect of LF Treatment on Growth of MALME-3MMelanomas

[0276] MALME-3M cells were injected subcutaneously (s.c.) into bothright and left dorsal upper flank areas of athymic nude mice. When tumorgrowth was established, the animals were randomized (n=5) based on thesize of the left-side tumor (˜70 mm³). Treatment began by directintratumoral (IT) injection at 2 day intervals only into right-side(“ipsilateral”) tumors of 10 μg PA+2 μg LF/mouse or 10 μg PA/mouse forcontrols. Contralateral (left-side) tumors were left untreated. Whenipsilateral tumors shrunk to a size that could not be visualized (in thePA+LF-10 treated group), at around day 10, the treatment route waschanged to ipsilateral s.c. injection approximately where the tumors hadbeen. Results are shown in FIG. 20A and 20B.

[0277] Ipsilateral MALME-3M tumors directly treated with PA+LFcompletely regressed after 6 doses. More importantly, the contralateraltumors also began to regress immediately. At the time the treatmentroute changed, contralateral tumors showed signs of regrowth but withcontinued treatment, began to regress again, whereas ipsilateral tumorsnever recurred. While each point is the mean of the group, analysis ofeach individual showed that two of the contralateral tumors continuouslyregressed, whereas one tumor grew back and the remaining two showedsigns of regrowth after the 16^(th) treatment. Treatment with PA alonehad no effect on tumor growth either ipsi- or contralaterally. Theseresults indicate that the antitumor effects of intratumoral/s.c. LFtreatment became systemic (presumably through distribution via bloodvessels, lymphatic networks, or both). A total of 19 LF doses weretolerated without any observable adverse effects on general health andbehavior of the animals.

EXAMPLE X Anti Tumor Effects of Infused Melanoma Cytotoxic Compositionin Human Patients

[0278] All patients treated have histologically confirmed melanoma andhave failed conventional therapy. Patients may be diagnosed as havingany stage of metastatic disease involving any organ system. Stagingdescribes both tumor and host, including organ of origin of the tumor,histologic type, histologic grade, extent of tumor size, site ofmetastases and functional status of the patient. A generalclassification includes the known ranges of Stage 1 (localized disease)to Stage 4 (widespread metastases). Patient history is obtained andphysical examination performed along with conventional tests ofcardiovascular and pulmonary function and appropriate radiologicprocedures. Histopathology is obtained to verify malignant disease.

Treatment Procedure

[0279] Doses of the test composition are determined as described aboveusing, inter alia, appropriate animal models of melanoma.

[0280] Two general classes of therapeutic compositions, described above,are administered:

[0281] (1) Proteins-MEK-proteases or functional derivatives;

[0282] (2) Small Molecule MEK inhibitors

[0283] A treatment consists of injecting the patient with 1, 100 or 1000μg of protein or polypeptide intravenously in 200 ml of normal salineover a one-hour period. Treatments are given 3×/week for a total of 12treatments. Patients with stable or regressing disease are treatedbeyond the 12th treatment. Treatment is given on either an outpatient orinpatient basis as needed.

Patient Evaluation

[0284] Assessment of response of the tumor to the therapy is made onceper week during therapy and 30 days thereafter. Depending on theresponse to treatment, side effects, and the health status of thepatient, treatment is terminated or prolonged from the standard protocolgiven above. Tumor response criteria are those established by theInternational Union Against Cancer and are listed below. RESPONSEDEFINITION Complete remission (CR) Disappearance of all evidence ofdisease Partial remission (PR) 50% decrease in the product of the twogreatest perpendicular tumor diameters; no new lesions Less than partialremission (<PR) 25%-50% decrease in tumor size, stable for at least 1month Stable disease <25% reduction in tumor size; no progression or newlesions Progression ≧25% increase in size of any one measured lesion orappearance of new lesions despite stabilization or remission of diseasein other measured sites

[0285] The efficacy of the therapy in a patient population is evaluatedusing conventional statistical methods, including, for example, the ChiSquare test or Fisher's exact test. Long-term changes in and short termchanges in measurements can be evaluated separately.

Results

[0286] One hundred and fifty patients are treated. The results aresummarized below. Positive tumor responses (at least partial remission)are observed in over 80% of the patients as follows: Response % PR 66%<PR 20% PR + <PR 86%

Toxicity

[0287] The incidence of side effects are between 10% and <1% of totaltreatments and include (in decreasing frequency): chills, fever; pain,nausea, respiratory, headache, tachycardia, vomiting, hypertension,hypotension, joint pain, rash, flushing, diarrhea, itching/hives, bloodynose, dizziness, cramps, fatigue, feeling faint, twitching, blurredvision, gastritis, redness on hand. Other minor changes observed areclinically insignificant.

[0288] The references cited above are all incorporated by referenceherein, whether specifically incorporated or not.

[0289] Having now fully described this invention, it will be appreciatedby those skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

What is claimed is:
 1. A method of killing melanoma cells comprisingcontacting said cells for an effective time with an effective amount ofan inhibitor of the MAPK pathway which induces apoptosis in said cells.2. The method of claim 1, wherein said inhibitor is a MEK-directedprotease.
 3. The method of claim 2, wherein said protease is Bacillusanthracis lethal factor or a functional derivative thereof.
 4. Themethod of claim 1 wherein said inhibitor is an organic small molecule.5. The method of claim 4 wherein said inhibitor is PD98059, U0126 orPD184352.
 6. The method of claim 1, wherein said contacting is in vivo.7. The method of claim 6 wherein said killing results in measurableregression of melanoma tumor or attenuation of melanoma growth.
 8. Amethod of protecting against melanoma in a susceptible subject,comprising administering to said subject that is (a) at risk fordevelopment of melanoma or, (b) in the case of an already treatedsubject, at risk for recurrence of melanoma, an effective amount of aMAPK-inhibitor.
 9. A method of inducing an antitumor response in amammal having melanoma, comprising administering an effective amount ofan inhibitor of the MAPK pathway to said mammal, which inhibitor iscytotoxic to melanoma cells, thereby inducing an antitumor response thatis (a) a partial antitumor response characterized by (i) at least a 50%decrease in the sum of the products of maximal perpendicular diametersof all measurable lesions; (ii) no evidence of new lesions, and (iii) noprogression of any preexisting lesions, or (b) a complete antitumorresponse characterized by the disappearance of all evidence of melanomadisease for at least one month.
 10. The method of claim 9 wherein saidantitumor response is a partial antitumor response.
 11. The method ofclaim 9, wherein said inhibitor is a MEK-directed protease.
 12. Themethod of claim 11, wherein said protease is Bacillus anthracis lethalfactor or a functional derivative thereof.
 13. The method of claim 9wherein said inhibitor is an organic small molecule.
 14. The method ofclaim 13 wherein said inhibitor is PD98059, U0126 or PD184352.
 15. Themethod of any of claims 9-14, wherein said mammal is a human.
 16. Amethod of inhibiting growth or recurrent growth of a melanoma tumor in amammal having melanoma or at risk for melanoma growth or recurrence,comprising administering an effective amount of an inhibitor of the MAPKpathway to said mammal, thereby inducing a cytotoxic response leading toapoptosis of melanoma cells in said mammal, which inhibits said growthor recurrent growth of said melanoma tumor.
 17. The method of claim 16wherein said inhibitor is a MEK-directed protease.
 18. The method ofclaim 17, wherein said protease is Bacillus anthracis lethal factor or afunctional derivative thereof.
 19. The method of claim 16 wherein saidinhibitor is an organic small molecule. 20 The method of claim 19wherein said inhibitor is PD98059, U0126 or PD184352.
 21. The method ofany of claims 16-20, wherein said mammal is a human.